Promoter Variants Of The Alpha-7 Nicotinic Acetylcholine Receptor

ABSTRACT

The present invention is directed to methods and compositions related to α7 acetylcholine nicotinic receptor genes, in particular, the human α7 nicotinic acetylcholine receptor gene. The human α7 nicotinic acetylcholine receptor gene is associated with the pathophysiological aspects of the disease schizophrenia. The present invention further provides methods and compositions to screen populations for abnormal α7 alleles, as well as methods and compositions for development of therapeutics.

This is a Continuation-In-Part of co-pending application Ser. No.10/723,940, filed Nov. 26, 2003 which is a Continuation-In-Part ofapplication Ser. No. 08/956,518, filed on Oct. 23, 1997 now issued asU.S. Pat. No. 6,875,606.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under NationalInstitutes of Health Grants DA09457, DA12231, AG00029, MH36321, andMH44212, and the Veterans Affairs Medical Research Service. TheGovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention is related to the alpha-7 neuronal nicotinicacetylcholine receptor gene. In particular, the present inventionprovides the human alpha-7 gene. In addition, the present inventionprovides methods and compositions for the diagnosis and treatment ofschizophrenia. Such compositions include, but are not limited to,polymorphisms within the human alpha-7 gene promoter core and/or 5′upstream regulatory regions.

BACKGROUND OF THE INVENTION

Schizophrenia is the most common chronic psychotic disorder of humans,affecting approximately one percent of the population worldwide (Eaton,Epidemiol Rev, 7:105, 1985). The mean lifetime risk of schizophrenia hasbeen estimated at one percent (Eaton, supra, 985). As the onset ofdisease usually occurs early in life, and results in serious chronicimpairment of cognition, behavior, and emotional state, schizophrenia isa major social problem in terms of cost, lost potential andproductivity, and family stress. Furthermore, estimates indicate thatthe mortality of schizophrenic patients is twice that of the generalpopulation (Tsuang et al., Arch Gen Psychiat, 36:1295, 1979). Themedical care of schizophrenic patients also presents a significantchallenge, as the patients are often unable to provide an accuratemedical history, and have difficulty complying with medical treatment.

The essential features of schizophrenia are the presence of psychoticsymptoms during some phase of the illness, a chronic course, anddeterioration in function. However, no combination of signs or symptomsis truly pathognomic of the disease. The DSM-IV criteria forschizophrenia (See, Hyman, “Schizophrenia,” in Dale and Federman (eds.),Scientific American Medicine, New York, N.Y., 13 VII: 1-5, 1994),requires a duration of at least six months, and a deterioration infunction. Psychotic symptoms typically exhibited by schizophreniapatients include disturbances in perception, abnormalities in thoughtcontent, and abnormalities in the form of thought. Perceptualdisturbances typically consist of hallucinations and illusions. Thecourse of schizophrenia is variable, although it is generallycharacterized by periods with exacerbation of psychotic symptoms,followed by periods of remission. Over a period of years, social andcognitive function usually deteriorates. Suicide attempts and depressionare common. As measured by frequency and severity of relapses,continuing symptoms, and overall functioning, approximately 80% ofschizophrenics have a poor outcome (Breier et al., Arch Gen Psychiat,48:239, 1991).

Although family, twin, and adoption studies indicate that schizophreniahas a significant genetic component, these studies also show that theinheritance of schizophrenia is complex, involving an uncertain mode oftransmission, incomplete penetrance, and probable genetic heterogeneity(Risch, Genet Epidemiol, 7:3, 1990; and Tsuang, Brit J Psychiat,163:299, 1993). Linkage studies using schizophrenia and relatedpsychiatric cases as phenotypes have found possible loci forschizophrenia at various chromosomal sites in subsets of families(Pulver et al., Am J Med Genet, 54:44, 1994; Coon et al., Am J MedGenet, 54:12, 1994; Wang et al., Nature Genet, 10:41, 1995; andSilverman et al., Am J Med Genet, 67:162, 1996). However, the findingsdo not account completely for the inheritance of schizophrenia, nor dothey delineate which aspects of this multifactoral illness might beinfluenced by a specific locus.

A variety of psychiatric disorders may mimic schizophrenia and thesymptoms of many disorders are similar. Thus, diagnosis has been basedon the course of illness (for example, acute onset and episodic coursein mania, compared with an insidious onset and chronic course inschizophrenia). In addition to schizophrenia, psychotic symptoms mayalso occur as a result of metabolic disturbances, structural brainlesions, other medical conditions, or drug toxicity. Thus, thedifferential diagnosis of schizophrenia must take into considerationsuch medical conditions as central nervous system neoplasm,hyperviscosity syndromes (i.e., due to hematologic malignancy),paraneoplastic syndromes, anoxia and postanoxic encephalopathy,hypertensive encephalopathy, AIDS encephalopathy, encephalitis,meningitis, brain abscess, Lyme disease, neurosyphilis, acuteintermittent porphyria, Addison's disease, Cushing's disease, hepaticencephalopathy, hypocalcemia, hypercalcemia, hypoglycemia,hypothyroidism, hyperthyroidism, Alzheimer's disease, complex partialseizures, Huntington's disease, multiple sclerosis, stroke, Wilson'sdisease, folic acid deficiency, pellagra, vitamin B₁₂ deficiency, andlupus cerebritis. Some drugs, such as alcohol, high-dose cocaine,high-dose amphetamines, marijuana, phencyclidine (PCP), hallucinogens,sedative-hypnotics, meperidine, non-steroidal anti-inflammatory drugs,pentazocine and other opiate mixed agonist-antagonists,anti-tuberculosis drugs (e.g., cycloserine, isoniazid, rifampin), otherantimicrobials, anticholinergic anti-parkinsonians, anti-histamines(e.g., diphenhydramine), atropine and derivatives, cyclicantidepressants, low-potency antipsychotic drugs (e.g., thioridazine andclozapine), meclizine, scopolamine, anti-arrhythmic (e.g., amiodarone,digitalis, and procainamide), captopril, amantadine, D₂ dopaminereceptor antagonists (e.g., bromocriptine, and pergolide), levodopa,estrogens, testosterone, glucocorticoids and adrenocorticotropic hormone(ACTH), thyroid replacement overdose, cimetidine, ranitidine,dextroamphetamine, methylphenidate, and over-the-counter decongestants(e.g., pseudoephedrine), diet pills, and pep pills, are commonlyassociated with psychotic symptoms.

Treatment of schizophrenic patients usually involves the use ofanti-psychotic drugs (e.g., haloperidol, haloperidol-like drugs, andatypical neuroleptics such as clozapine), maintenance of a safe,predictable environment, and supportive psychotherapy to improve socialand coping skills of patients. Stress reduction also appears to preventrelapses. While these drugs are useful in treating the symptoms ofschizophrenia, there are also problems associated with their use. Forexample, the use of clozapine is complicated by the idiosyncraticoccurrence of agranulocytosis, necessitating weekly monitoring of thewhite blood cell counts of patients taking this drug (See, Hyman, supra,1994).

Despite advances in treatment and diagnostic methods, there remains aneed for methods to diagnose and treat schizophrenic patients. Indeed,methods to diagnose and screen large populations for geneticcomponent(s) associated with schizophrenia, as well as other psychosesare needed in order to provide reliable diagnoses that are not dependentupon the multifactoral criteria presently in use. Improved methods oftreatment are also needed, including drugs and other therapeutics thatdo not have the side effects and other undesirable properties associatedwith the currently used drugs.

SUMMARY OF THE INVENTION

The present invention is related to the alpha-7 neuronal nicotinicacetylcholine receptor gene. In particular, the present inventionprovides the human alpha-7 gene. In addition, the present inventionprovides methods and compositions for the diagnosis and treatment ofschizophrenia. Such compositions include, but are not limited to,polymorphisms within the human alpha-7 gene promoter core and/or 5′upstream regulatory regions.

In one embodiment, the present invention contemplates a compositioncomprising an isolated 5′ upstream regulatory nucleotide sequence of ahuman alpha-7 nicotinic receptor, wherein the nucleotide sequencecomprises at least one polymorphism. In one embodiment, the sequencecomprises a portion of SEQ ID NO:181. In one embodiment, the at leastone polymorphism comprises one or more of those listed in Table 23. Inone embodiment, the polymorphism is rs3087454 (−1831 bp).

In one embodiment, the present invention contemplates a compositioncomprising an isolated regulatory binding site sequence encoded by anisolated 5′ upstream regulatory nucleotide sequence of a human alpha-7nicotinic receptor, wherein the nucleotide sequence comprises at leastone polymorphism. In one embodiment, the sequence comprises a portion ofSEQ ID NO:181. In one embodiment, the at least one polymorphismcomprises one or more of those listed in Table 23. In one embodiment,the polymorphism is rs3087454 (−1831 bp).

In one embodiment, the present invention contemplates a method ofidentifying individuals predisposed to schizophrenia comprising:providing a nucleic acid from a human subject; wherein the nucleic acidcomprises an α7 regulatory allele; detecting the presence of at leastone polymorphism within the α7 regulatory allele; and correlating thepresence of the at least one polymorphism with a predisposition toschizophrenia. In some embodiments, the at least one polymorphismcomprises one or more of those listed in Table 23. In one embodiment,the polymorphism is rs3087454 (−1831 bp). In other embodiments theallele comprises two or more polymorphisms. In one embodiment, the atleast one polymorphism comprises a promoter polymorphism thatcontributes to reduced α7 regulatory protein transcription. In oneembodiment, the detecting comprises at least one technique selected fromthe group including, but not limited to, polymerase chain reaction,heteroduplex analysis, single stand conformational polymorphismanalysis, denaturing high performance liquid chromatography, ligasechain reaction, comparative genome hybridization, Southern blotting ornucleic acid sequencing. In some embodiments, the nucleic acid from thesubject is derived from a sample selected from the group including, butnot limited to, a biopsy material or blood. In one embodiment, themethod further comprises step d) providing a diagnosis to the subjectbased on the presence or absence of the at least one polymorphism. Inone embodiment, the diagnosis differentiates schizophrenia from otherforms of mental illness.

In one embodiment, the present invention contemplates a method,comprising: a) providing; i) a biological sample suspected of containinga first polynucleotide encoding an α7 regulatory protein; ii) a secondpolynucleotide comprising at least a portion of SEQ ID NO: 181, whereinsaid portion is capable of hybridizing to said first polynucleotide; b)hybridizing said first polynucleotide to said second polynucleotide toproduce a hybridization complex; c) detecting an α7 nicotinic acidregulatory allele within said first polynucleotide, wherein said allelecomprises at least one polymorphism. In one embodiment, the at least onepolymorphism is rs3087454 (−1831 bp). In one embodiment, the at leastone polymorphism comprises one or more of those listed in Table 23. Inone embodiment, the method further comprises step (d) detecting saidhybridization complex, wherein said complex correlates with said firstpolynucleotide. In one embodiment, the biological sample is selectedfrom the group consisting of brain tissue and blood. In one embodiment,the biological sample is from a human. In one embodiment, the human issuspected of suffering from a condition selected from the groupincluding, but not limited to, schizophrenia, small cell lung carcinoma,breast cancer, or nicotine-dependent illness. In one embodiment, themethod further comprises, before step (b) amplifying said firstpolynucleotide by polymerase chain reaction.

In one embodiment, the present invention contemplates a compositioncomprising a vector comprising a 5′ upstream regulatory nucleotidesequence of a human alpha-7 nicotinic receptor, wherein said sequencecomprises at least one polymorphism. In one embodiment, the nucleotidesequence comprises at least a portion of SEQ ID NO:181. In oneembodiment, the polymorphism is rs3087454 (−1831 bp). In one embodiment,the at least one polymorphism comprises one or more of those listed inTable 23.

In one embodiment, the present invention contemplates a compositioncomprising a host cell transformed with a vector comprising a 5′upstream regulatory nucleotide sequence of a human alpha-7 nicotinicreceptor, wherein the nucleotide sequence comprises a polymorphism. Inone embodiment, the nucleotide sequence comprises at least a portion ofSEQ ID NO:181. In one embodiment, the polymorphism is rs3087454 (−1831bp). In one embodiment, the at least one polymorphism comprises one ormore of those listed in Table 23. In one embodiment, the host cell isselected from the group including, but not limited to, bacteria, yeast,amphibian, or mammalian cells. In one embodiment, the host cell is ahuman cell. In one embodiment, the host cell is a cell line. In oneembodiment, the host cell is contained within an animal.

In one embodiment, the present invention contemplates a compositioncomprising hybridization complex, wherein the complex comprises a firstpolynucleotide sequence comprising at least fifteen nucleotides, whereinsaid first polynucleotide is hybridized, under stringent conditions, toat least a portion of a second polynucleotide sequence, wherein thesecond polynucleotide sequence comprises at least a portion of SEQ IDNO:181 and at least one polymorphism. In one embodiment, thepolymorphism is rs3087454 (−1831 bp). In one embodiment, the at leastone polymorphism comprises one or more of those listed in Table 23.

In one embodiment, the present invention contemplates a method foramplifying a nucleic acid derived from a biological sample comprising;providing the biological sample is suspected of containing nucleic acidencoding an alpha-7 nicotinic acid gene regulatory protein and a testsample suspected of containing amplifiable nucleic acid encoding thealpha-7 regulatory protein; isolating the amplifiable nucleic acid fromthe test sample; combining the amplifiable nucleic acid withamplification reagents, and at least two primers selected from the groupcomprising SEQ ID NO:182, SEQ ID NO: 183, and primers having the nucleicacid sequence set forth in FIG. 20 to form a reaction mixture; andcombining the reaction mixture with an amplification enzyme underconditions wherein the amplifiable nucleic acid is amplified to formamplification product. In one embodiment, the method further comprisesthe step of detecting the amplification product.

In one embodiment, the method further comprises the step of detectingthe amplification product by hybridizing the amplification product witha probe having a nucleic acid sequence is selected from a groupcomprising SEQ ID NO:182, SEQ ID NO: 183, or the sequences set forth inFIG. 20. In one embodiment, the test sample is a sample selected fromthe group including, but not limited to, brain tissue or blood. In oneembodiment, the test sample is from a human. In one embodiment, thehuman is suspected of suffering from a condition selected from the groupincluding, but not limited to, schizophrenia, small cell lung carcinoma,breast cancer, and nicotine-dependent illness.

In one embodiment, the present invention contemplates a method foramplifying a nucleic acid from a sample suspected of containing nucleicacid encoding an alpha-7 nicotinic acid receptor gene regulatory proteincomprising the steps of: providing a test sample suspected of containingamplifiable nucleic acid encoding the alpha-7 regulatory protein;isolating the amplifiable nucleic acid from the test sample; combiningthe amplifiable nucleic acid with amplification reagents, and a firstprimer set comprising at least two primers selected from the groupconsisting of SEQ ID NO: 182, SEQ ID NO: 183, and those set forth inFIG. 20, to form a first reaction mixture; combining the reactionmixture with an amplification enzyme under conditions wherein theamplifiable nucleic acid is amplified to form a first amplificationproduct; combining the first reaction mixture with amplificationreagents, and a second primer set comprising at least two primersselected from the group consisting of the sequences set forth in Table20, to form a second reaction mixture; combining the second reactionmixture with an amplification enzyme under conditions wherein theamplifiable nucleic acid is amplified to form a second amplificationproduct; and detecting the first or second amplification product. In oneembodiment, the method further comprises the step of detecting theamplification product by hybridizing the amplification product with aprobe having a nucleic acid sequence selected from the group consistingof the nucleic acid sequence set forth in FIG. 20. In one embodiment,the test sample is a sample selected from the group including, but notlimited to, brain tissue or blood. In one embodiment, the test sample isderived from a human. In one embodiment, the human is suspected ofsuffering from a condition selected from the group including, but notlimited to, schizophrenia, small cell lung carcinoma, breast cancer, andnicotine-dependent illness.

In one embodiment, the present invention contemplates a kit fordetermining if a subject is predisposed to schizophrenia, comprising: atleast one reagent suitable for use in specifically detecting at leastone polymorphism in an α7 nicotinic acid receptor regulatory allele; andinstructions for determining whether a subject is predisposed toschizophrenia. In some embodiments, the at least one polymorphismcomprises one or more of those listed in Table 23. In preferredembodiments, the at least one polymorphism comprises a promoterpolymorphism that contributes to reduced α7 regulatory proteintranscription. In one embodiment, the at least one reagent comprises anucleic acid probe that hybridizes under stringent conditions to anucleic acid sequence selected from the group including, but not limitedto, the coding strand of the α7 regulatory allele, or the noncodingstrand of the α7 regulatory allele. In one embodiment, the at least onereagent comprises a sense primer and an antisense primer flanking the atleast one polymorphism in the α7 regulatory allele. In one embodiment,at least one of the primers comprises a fluorescent tag. In oneembodiment, the instructions comprise instructions required by theUnited States Food and Drug Administration for use in in vitrodiagnostic products.

In one embodiment, the present invention contemplates a method ofscreening compounds, comprising: providing: i) at least one cellcomprising an α7 nicotinic acid receptor regulatory allele with at leastone polymorphism, and ii) one or more test compounds; and contacting theat least one cell with the test compound; and detecting a change in α7regulatory allele expression in the at least one cell in the presence ofthe test compound relative to the absence of the test compound. In oneembodiment, the detecting comprises detecting α7 regulatory allele mRNA.In one embodiment, the detecting comprises detecting α7 regulatoryprotein. In one embodiment, the cell is a neuroblastoma cell. In oneembodiment, the test compound comprises a drug. In one embodiment, theat least one polymorphism comprises a promoter polymorphism thatcontributes to reduced α7 transcription.

In one embodiment, the present invention contemplates a method ofscreening compounds, comprising: providing: i) at least one cellcomprising an α7 nicotinic acid receptor promoter in operablecombination with a reporter gene, wherein said α7 promoter comprises aregulatory allele having at least one polymorphism, and ii) one or moretest compounds; and contacting the at least one cell with the testcompound; and detecting a change in expression of the reporter gene inthe at least one cell in the presence of the test compound relative tothe absence of the test compound. In one embodiment, the detectingcomprises detecting reporter gene mRNA or polypeptide. In oneembodiment, the detecting comprises detecting reporter gene function. Inone embodiment, the cell is a neuroblastoma cell. In one embodiment, thetest compound comprises a drug. In one embodiment, the reporter gene isthe firefly luciferase gene.

In one embodiment, the present invention contemplates a method ofidentifying individuals predisposed to schizophrenia, comprising:providing a nucleic acid sample from a subject, the sample containing anα7 nicotinic acid receptor regulatory allele; correlating the identityof the α7 regulatory allele with a predisposition to schizophrenia. Inone embodiment, the identity of the α7 regulatory allele is determinedusing at least one technique selected from the group including, but notlimited to, polymerase chain reaction, heteroduplex analysis, singlestrand conformational polymorphism analysis, denaturing high performanceliquid chromatography, ligase chain reaction, comparative genomehybridisation, Southern blotting or nucleic acid sequencing. In oneembodiment, the nucleic acid sample from the subject is selected fromthe group including, but not limited to, a biopsy material or blood. Inone embodiment, the method further comprises a step providing adiagnosis to the subject based on the identity of the α7 regulatoryallele.

In one embodiment, the present invention contemplates a method forproducing anti-α7 nicotinic acid receptor regulatory protein antibodies,comprising, exposing an animal having immunocompetent cells to animmunogen comprising at least an antigenic portion of α7 regulatoryprotein, under conditions such that immunocompetent cells produceantibodies directed against the portion of α7 regulatory protein. In oneembodiment, the α7 regulatory binding site has affinity for a α7regulatory peptide or protein. In one embodiment, the method furthercomprises the step of harvesting the antibodies. In one embodiment, themethod further comprises the step of fusing the immunocompetent cellswith an immortal cell line under conditions such that a hybridoma isproduced. In one embodiment, the immunogen comprises a fusion protein.

In one embodiment, the present invention contemplates a method fordetecting abnormal α7 nicotinic acid receptor regulatory gene expressioncomprising the steps of: a) providing a biological sample suspected ofcontaining a test α7 regulatory gene; and a control containing aquantitated α7 regulatory gene; and b) comparing the test α7 regulatorygene in the sample with the quantitated α7 regulatory gene in thecontrol to determine the relative concentration of the test α7regulatory gene in the sample. In one embodiment, the control contains ahigher concentration of quantitated α7 regulatory gene than theconcentration of the test α7 regulatory gene in the sample. In oneembodiment, the biological sample comprises a patient sample, whereinsaid patient sample comprises reduced α7 regulatory protein. In oneembodiment, the sample contains a normal amount of the α7 regulatoryprotein. In one embodiment, the comparing comprises a detection methodselected from the group including, but not limited to, Western blotanalysis, Northern blot analysis, Southern blot analysis, denaturingpolyacrylamide gel electrophoresis, reverse transcriptase-coupledpolymerase chain reaction, enzyme-linked immunosorbent assay,radioimmunoassay, or fluorescent immunoassay. In one embodiment, themethod detects α7 regulatory gene or mRNA in the genome of the animalsource of the biological sample. In one embodiment, the method detectsthe expression of α7 regulatory protein in the genome of the animalsource of the biological sample. In one embodiment, the method detectsthe presence of abnormal or mutated α7 regulatory proteins or genesequences in the biological sample.

In one embodiment, the present invention provides an isolated nucleotidesequence encoding at least a portion of the human alpha-7 nicotinicreceptor, wherein the sequence is selected from the group consisting ofSEQ ID NOS:84-103. In an alternative embodiment, the present inventionprovides an isolated regulatory peptide sequence that binds to theisolated nucleotide sequence, wherein the nucleotide sequence isselected from the group consisting of SEQ ID NOS:84-103. In anotherembodiment, the nucleotide sequence further comprises 5′ and 3′ flankingregions. In yet another embodiment, the nucleotide sequence furthercomprises intervening regions. In a further embodiment, the presentinvention provides an isolated polynucleotide sequence comprising acombination of two or more nucleotide sequences, wherein the nucleotidesequences are selected from the group consisting of SEQ ID NOS:84-103.It is not intended that the combination comprise any particular numberor order of these nucleotide sequences, nor is it intended that thecombination be limited to the inclusion of any particular nucleotidesequence.

In another embodiment, the present invention provides vectors comprisinga nucleotide sequence, wherein the nucleotide sequence comprises atleast one nucleotide sequence selected from the group consisting of SEQID NOS:84-103. In another embodiment, the present invention provides ahost cell transformed with a vector comprising a nucleotide sequence,wherein the nucleotide sequence comprises at least one nucleotidesequence selected from the group consisting of SEQ ID NOS:84-103. In oneembodiment, the host cell is selected from the group consisting ofbacteria, yeast, amphibian, and mammalian cells. In one preferredembodiment, the host cell is a human cell. In an alternative preferredembodiment, the host cell is a cell line, while in another preferredembodiment, the host cell is contained within an animal.

The present invention also provides a first polynucleotide sequencecomprising at least fifteen nucleotides, which hybridizes understringent conditions to at least a portion of a second polynucleotidesequence, wherein the second polynucleotide sequence is selected fromthe polynucleotide sequences selected from the group consisting of SEQID NOS:84-103.

The present invention also provides methods for detection of apolynucleotide encoding alpha-7 protein in a biological sample suspectedof containing the polynucleotide encoding alpha-7, comprising the stepof hybridizing at least a portion of a polynucleotide sequence selectedfrom the group consisting of SEQ ID NOS:9-11, and 84-103, to nucleicacid of the biological sample to produce an hybridization complex. Inone embodiment, the method further comprises the step of detecting thehybridization complex, wherein the presence of the complex correlateswith the presence of a polynucleotide encoding alpha-7 in the biologicalsample. In another embodiment, the biological sample is a sampleselected from the group consisting of brain tissue and blood. In onepreferred embodiment, the biological sample is from a human. In yetanother embodiment, the human is suspected of suffering from a conditionselected from the group consisting of schizophrenia, small cell lungcarcinoma, breast cancer, and nicotine-dependent illness. In yet anotherpreferred embodiment of the method, the nucleic acid of the biologicalsample is amplified by the polymerase chain reaction prior tohybridization.

The present invention also provides methods for amplification of nucleicacid from a sample suspected of containing nucleic acid encodingalpha-7, comprising the steps of: providing a test sample suspected ofcontaining amplifiable nucleic acid encoding alpha-7; isolating theamplifiable nucleic acid from the test sample; combining the amplifiablenucleic acid with amplification reagents, and at least two primersselected from the group consisting of primers having the nucleic acidsequence set forth in SEQ ID NOS:1-8, and 12-83 to form a reactionmixture; and combining the reaction mixture with an amplification enzymeunder conditions wherein the amplifiable nucleic acid is amplified toform amplification product. In one embodiment, the method furthercomprises the step of detecting the amplification product. In analternative embodiment, the detecting is accomplished by hybridizationof the amplification product with a probe having the nucleic acidsequence is selected from group of the sequences set forth in SEQ IDNO:9-11. In one preferred embodiment, the test sample is a sampleselected from the group consisting of brain tissue and blood. In analternative preferred embodiment, the test sample is from a human. Inyet another embodiment, the human is suspected of suffering from acondition selected from the group consisting of schizophrenia, smallcell lung carcinoma, breast cancer, and nicotine-dependent illness.

The present invention also provides methods for amplification of nucleicacid from a sample suspected of containing nucleic acid encoding alpha-7comprising the steps of: providing a test sample suspected of containingamplifiable nucleic acid encoding alpha-7; isolating the amplifiablenucleic acid from the test sample; combining the amplifiable nucleicacid with amplification reagents, and a first primer set comprising atleast two primers selected from the group consisting of the sequencesset forth in SEQ ID NOS:65-70, to form a first reaction mixture;combining the reaction mixture with an amplification enzyme underconditions wherein the amplifiable nucleic acid is amplified to form afirst amplification product; combining the first reaction mixture withamplification reagents, and a second primer set comprising at least twoprimers selected from the group consisting of the sequences set forth inSEQ ID NOS:57-59, 61, 63, 67, and 73-75, to form a second reactionmixture; combining the second reaction mixture with an amplificationenzyme under conditions wherein the amplifiable nucleic acid isamplified to form a second amplification product; and detecting thefirst or second amplification product.

In one preferred embodiment of the method, the detecting compriseshybridizing the amplification product with a probe having a nucleic acidsequence selected from the group consisting of the nucleic acid sequenceset forth in SEQ ID NOS:9-11. In yet another embodiment, the test sampleis a sample selected from the group consisting of brain tissue andblood. In another preferred embodiment of the method, the test sample isfrom a human. In a further embodiment, the human is suspected ofsuffering from a condition selected from the group consisting ofschizophrenia, small cell lung carcinoma, breast cancer, andnicotine-dependent illness.

Additionally, the present invention provides methods of identifyingindividuals predisposed schizophrenia comprising: providing a nucleicacid from a human subject; wherein the nucleic acid comprises an α7allele; detecting the presence of at least one polymorphism within theα7 allele; and correlating the presence of the at least one polymorphismwith a predisposition to schizophrenia. In some embodiments the at leastone polymorphism comprises one or more of a −241 A to G substitution, a−194 G to C substitution, a −191 G to A substitution, a −190 Ginsertion, a −180 G to C substitution, a −178 CGGGGG insertion, a −178 Gdeletion, a −166 C to T substitution, a −143 G to A substitution, a −140CGGG insertion, a −93 C to G substitution, a −92 G to A substitution, a−86 C to T substitution, and a 46 G to T substitution. In otherembodiments the at least one polymorphism comprises two or morepolymorphisms. In some preferred embodiments, the at least onepolymorphism comprises a promoter polymorphism that contributes toreduced α7 transcription. The present invention provides methods whereinthe detecting step is accomplished using at least one technique selectedfrom the group consisting of polymerase chain reaction, heteroduplexanalysis, single stand conformational polymorphism analysis, denaturinghigh performance liquid chromatography, ligase chain reaction,comparative genome hybridisation, Southern blotting and sequencing. Insome embodiments, the nucleic acid from the subject is derived from asample selected from the group consisting of a biopsy material andblood. Moreover embodiments are provided which further comprise step d)providing a diagnosis to the subject based on the presence or absence ofthe at least one polymorphism. In preferred embodiments, the diagnosisdifferentiates schizophrenia from other forms of mental illness.

The present invention also provides kits for determining if a subject ispredisposed to schizophrenia, comprising: at least one reagent suitablefor use in specifically detecting at least one polymorphism in an α7allele; and instructions for determining whether a subject ispredisposed to schizophrenia. In some embodiments, the at least onepolymorphism comprises one or more of a −241 A to G substitution, a −194G to C substitution, a −191 G to A substitution, a −190 G insertion, a−180 G to C substitution, a −178 CGGGGG insertion, a −178 G deletion, a−166 C to T substitution, a −143 G to A substitution, a −140 CGGGinsertion, a −93 C to G substitution, a −92 G to A substitution, a −86 Cto T substitution, and a −46 G to T substitution. In preferredembodiments, the at least one polymorphism comprises a promoterpolymorphism that contributes to reduced α7 transcription. The presentinvention further provides embodiments in which the at least one reagentcomprises a nucleic acid probe that hybridizes under stringentconditions to a nucleic acid sequence selected from the group consistingof the coding strand of the α7 gene, and the noncoding strand of the α7gene. In some preferred embodiments, the at least one reagent comprisesa sense primer and an antisense primer flanking the at least onepolymorphism in the α7 allele. In a subset of these, at least one of theprimers comprises a fluorescent tag. Moreover, in some embodiments, theinstructions comprise instructions required by the United States Foodand Drug Administration for use in in vitro diagnostic products.

Also provided by the present invention are methods of screeningcompounds, comprising: providing: i) at least one cell comprising an α7allele with at least one polymorphism, and ii) one or more testcompounds; and contacting the at least one cell with the test compound;and detecting a change in α7 expression in the at least one cell in thepresence of the test compound relative to the absence of the testcompound. In some embodiments the detecting comprises detecting α7 mRNA,while in others the detecting comprises detecting α7 polypeptide. Inpreferred embodiments, the cell is a neuroblastoma cell. In otherpreferred embodiments, the test compound comprises a drug. Moreover, inparticularly preferred embodiments, the at least one polymorphismcomprises a promoter polymorphism that contributes to reduced α7transcription.

In alternative embodiments, the present invention provides methods ofscreening compounds, comprising: providing: i) at least one cellcomprising an α7 promoter in operable combination with a reporter gene,wherein said α7 promoter comprises at least one polymorphism, and ii)one or more test compounds; and contacting the at least one cell withthe test compound; and detecting a change in expression of the reportergene in the at least one cell in the presence of the test compoundrelative to the absence of the test compound. In some embodiments thedetecting comprises detecting reporter gene mRNA or polypeptide, whilein others the detecting comprises detecting reporter gene function. Inpreferred embodiments, the cell is a neuroblastoma cell. In otherpreferred embodiments, the test compound comprises a drug. In anexemplary embodiment, the reporter gene is the firefly luciferase gene.

Furthermore, the present invention provides methods of identifyingindividuals predisposed to schizophrenia, comprising: providing anucleic acid sample from a subject, the sample containing an α7 allele;correlating the identity of the α7 allele with a predisposition toschizophrenia. In some embodiments, the identity of the α7 allele isdetermined using at least one technique selected from the groupconsisting of polymerase chain reaction, heteroduplex analysis, singlestand conformational polymorphism analysis, denaturing high performanceliquid chromatography, ligase chain reaction, comparative genomehybridisation, Southern blotting and sequencing. In preferredembodiments, the nucleic acid sample from the subject is selected fromthe group consisting of a biopsy material and blood. Moreoverembodiments are provided which further comprise step c) providing adiagnosis to the subject based on the identity of the α7 allele.

The present invention also provides methods for producing anti-α7antibodies (including, but not limited to antibodies directed againstpeptides comprising α7), comprising, exposing an animal havingimmunocompetent cells to an immunogen comprising at least an antigenicportion of α7 protein, under conditions such that immunocompetent cellsproduce antibodies directed against the portion of α7. In preferredembodiments, the α7 peptide or protein is human α7. In one embodiment,the method further comprises the step of harvesting the antibodies. Inan alternative embodiment, the method comprises the step of fusing theimmunocompetent cells with an immortal cell line under conditions suchthat a hybridoma is produced. In other embodiments, the immunogencomprises a fusion protein.

The present invention also provides methods for detecting abnormal α7expression comprising the steps of: a) providing a sample suspected ofcontaining test α7; and a control containing a quantitated α7; and b)comparing the test α7 in the sample with the quantitated α7 in thecontrol to determine the relative concentration of the test α7 in thesample. In one embodiment of the method, the control contains a higherconcentration of quantitated α7 than the concentration of the test α7 inthe sample. Thus, the methods are capable of identifying samples (e.g.,patient samples) with reduced α7 protein. The methods also provide meansto detect samples that contain a normal amount of the α7 protein. Inaddition, the methods may be conducted using any suitable means todetermine the relative concentration of α7 in the test and controlsamples, including but not limited to the means selected from the groupconsisting of Western blot analysis, Northern blot analysis, Southernblot analysis, denaturing polyacrylamide gel electrophoresis, reversetranscriptase-coupled polymerase chain reaction, enzyme-linkedimmunosorbent assay, radioimmunoassay, and fluorescent immunoassay.Thus, the methods may be conducted to determine the presence of α7 inthe genome of the animal source of the test sample, or the expression ofα7 (mRNA or protein), as well as detect the presence of abnormal ormutated α7 proteins or gene sequences in the test samples.

In one preferred embodiment, the presence of α7 is detected byimmunochemical analysis. For example, the immunochemical analysis cancomprise detecting binding of an antibody specific for an epitope of α7.In another preferred embodiment of the method, the antibody comprisespolyclonal antibodies, while in another preferred embodiment, theantibody is comprises monoclonal antibodies.

The antibodies used in the methods invention may be prepared usingvarious immunogens. In one embodiment, the immunogen is a human α7peptide to generate antibodies that recognize human α7. Such antibodiesinclude, but are not limited to polyclonal, monoclonal, chimeric, singlechain, Fab fragments, and a Fab expression library.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to α7 (e.g., human α7). For the production ofantibody, various host animals can be immunized by injection with thepeptide corresponding to the human α7 epitope including but not limitedto rabbits, mice, rats, sheep, goats, etc. In a preferred embodiment,the peptide is conjugated to an immunogenic carrier (e.g., diphtheriatoxoid; bovine serum albumin, BSA; or keyhole limpet hemocyanin, KLH).Various adjuvants may be used to increase the immunological response,depending on the host species, including but not limited to Freund's(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,dinitrophenol, and potentially useful human adjuvants such as BCG(Bacille Calmette-Guerin) and Corynebacterium parvum.

For preparation of monoclonal antibodies directed against α7, anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture may be used (See, e.g., Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.). These include but are not limited to:the hybridoma technique originally developed by Köhler and Milstein(Köhler and Milstein, Nature 256:495-497, 1975); the trioma technique;the human B-cell hybridoma technique (See e.g., Kozbor et al., ImmunolToday, 4:72, 1983); and the EBV-hybridoma technique (Cole et al., inMonoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96,1985).

In an additional embodiment of the invention, monoclonal antibodies canbe produced in germ-free animals utilizing recent technology (See e.g.,PCT/US90/02545). According to the invention, human antibodies may beused and can be obtained by using human hybridomas (Cote et al., ProcNatl Acad Sci USA, 80:2026-2030, 1983), or by transforming human B cellswith EBV virus in vitro (Cole et al., supra, 1985).

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778; herein incorporated byreference) can be adapted to produce α7 single chain antibodies. Anadditional embodiment of the invention utilizes the techniques describedfor the construction of Fab expression libraries (Huse et al., Science,246:1275-1281, 1989) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity for α7.

Antibody fragments that contain the idiotype (antigen binding region) ofthe antibody molecule can be generated by known techniques. For example,such fragments include but are not limited to: the F(ab′)2 fragmentwhich can be produced by pepsin digestion of the antibody molecule; theFab′ fragments which can be generated by reducing the disulfide bridgesof the F(ab′)2 fragment, and the Fab fragments which can be generated bytreating the antibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art including but not limitedto radioimmunoassay, enzyme-linked immunosorbent assay, “sandwich”immunoassay, gel diffusion precipitin reactions, immunodiffusion assays,in situ immunoassays (using colloidal gold, enzyme or radioisotopelabels, for example), Western Blots, precipitation reactions,agglutination assays (e.g., gel agglutination assays, hemagglutinationassays, etc.), complement fixation assays, immunofluorescence assays,protein A assays, and immunoelectrophoresis assays, etc.

In one embodiment, antibody binding is detected by detecting a label onthe primary antibody. In another embodiment, the primary antibody isdetected by detecting binding of a secondary antibody or reagent to theprimary antibody. In a further embodiment, the secondary antibody islabeled. Many means are known in the art for detecting binding in animmunoassay and are within the scope of the present invention. (As iswell known in the art, the immunogenic peptide should be provided freeof the carrier molecule used in any immunization protocol. For example,if the peptide was conjugated to KLH, it may be conjugated to BSA, orused directly, in a screening assay.)

The foregoing antibodies can be used in methods known in the artrelating to the localization and structure of α7 (e.g., for Westernblotting), measuring levels thereof in appropriate biological samples,etc. The antibodies can be used to detect α7 in a biological sample froman individual. The biological sample can be a biological fluid, such asbut not limited to, blood, serum, plasma, cerebrospinal fluid (CSF), andthe like, containing cells. In particular, α7 can be detected fromcellular sources, such as, but not limited to, brain tissue.

The biological samples can then be tested directly for the presence ofα7 using an appropriate strategy (e.g., ELISA or radioimmunoassay) andformat (e.g., microwells, dipstick for instance as described inInternational Patent Publication WO 93/03367, etc.). Alternatively,proteins in the sample can be size separated (e.g., by polyacrylamidegel electrophoresis, PAGE, in the presence or absence of sodium dodecylsulfate, SDS), and the presence of α7 detected by immunoblotting(Western blotting). Immunoblotting techniques are generally moreeffective with antibodies generated against a peptide corresponding toan epitope of a protein, and hence, are particularly suited to thepresent invention.

The foregoing explanations of particular assay systems are presentedherein for purposes of illustration only, in fulfillment of the duty topresent an enabling disclosure of the invention. It is to be understoodthat the present invention contemplates a variety of immunochemicalassay protocols within its spirit and scope.

In some preferred aspects, genomic DNA or mRNA is amplified by PCR, andthe amplified DNA is tested for the presence of mutation(s). PCRamplification is well known in the art (Cameron et al., Science,257:383-387, 1992; Saksela et al., Proc Natl Acad Sci USA, 91:1104-1108,1994). For example, mRNA can be detected by reversetranscriptase-initiated PCR (See, e.g., Saksela et al., J Virol,67:7423-27, 1993). PCR can be carried out (e.g., by use of aPerkin-Elmer Cetus thermal cycler and Taq polymerase, e.g., Gene Amp®,Boehringer Mannheim). The amplified PCR products can be analyzed byimmobilization on membranes and hybridization with specificoligonucleotide probes, or by treatment with specific endonucleases andanalysis of the products by gel electrophoresis. Labeling of the cleavedPCR products can be accomplished by incorporation of radiolabelednucleotides, endlabeling (e.g., with ³²P-ATP), or by staining withethidium bromide.

The present invention also provides methods and compositions suitablefor gene therapy for individuals deficient in α7 expression, production,or function. Viral vectors commonly used for in vivo or ex vivotargeting and therapy procedures are DNA-based vectors and retroviralvectors. Methods for constructing and using viral vectors are known inthe art (See, e.g., Miller and Rosman, BioTechn, 7:980-990, 1992).Preferably, the viral vectors are replication defective, that is, theyare unable to replicate autonomously in the target cell. In general, thegenome of the replication defective viral vectors which are used withinthe scope of the present invention lack at least one region which isnecessary for the replication of the virus in the infected cell. Theseregions can either be eliminated (in whole or in part), be renderednon-functional by any technique known to a person skilled in the art.These techniques include the total removal, substitution (by othersequences, in particular by the inserted nucleic acid), partial deletionor addition of one or more bases to an essential (for replication)region. Such techniques may be performed in vitro (i.e., on the isolatedDNA) or in situ, using the techniques of genetic manipulation or bytreatment with mutagenic agents.

Preferably, the replication defective virus retains the sequences of itsgenome, which are necessary for encapsidating the viral particles. DNAviral vectors include an attenuated or defective DNA virus, including,but not limited to, herpes simplex virus (HSV), papillomavirus, EpsteinBarr virus (EBV), adenovirus, adeno-associated virus (AAV), and thelike. Defective viruses, which entirely or almost entirely lack viralgenes, are preferred, as defective virus is not infective afterintroduction into a cell. Use of defective viral vectors allows foradministration to cells in a specific, localized area, without concernthat the vector can infect other cells. Thus, a specific tissue can bespecifically targeted. Examples of particular vectors include, but arenot limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt etal., Mol. Cell. Neurosci., 2:320-330, 1991), defective herpes virusvector lacking a glycoprotein L gene (See e.g., Patent Publication RD371005 A), or other defective herpes virus vectors (See e.g.,International Patent Publication No. WO 94/21807; and InternationalPatent Publication No. WO 92/05263); an attenuated adenovirus vector,such as the vector described (Stratford-Perricaudet et al., J ClinInvest, 90:626-630, 1992; and La Salle et al., Science, 259:988-990,1993); and a defective adeno-associated virus vector (Samulski et al., JVirol, 61:3096-3101, 1987; Samulski et al., J Virol, 63:3822-3828, 1989;and Lebkowski et al., Mol Cell Biol, 8:3988-3996, 1988).

Preferably, for in vivo administration, an appropriate immunosuppressivetreatment is employed in conjunction with the viral vector (e.g.,adenovirus vector), to avoid immuno-deactivation of the viral vector andtransfected cells. For example, immunosuppressive cytokines, such asinterleukin-12 (IL-12), interferon-gamma (IFN-γ), or anti-CD4 antibody,can be administered to block humoral or cellular immune responses to theviral vectors. In addition, it is advantageous to employ a viral vectorthat is engineered to express a minimal number of antigens.

In a preferred embodiment, the vector is an adenovirus vector.Adenoviruses are eukaryotic DNA viruses that can be modified toefficiently deliver a nucleic acid of the invention to a variety of celltypes. Various serotypes of adenovirus exist. Of these serotypes,preference is given, within the scope of the present invention, to type2 or type 5 human adenoviruses (Ad 2 or Ad 5), or adenoviruses of animalorigin (See, WO94/26914). Those adenoviruses of animal origin, which canbe used within the scope of the present invention, include adenovirusesof canine, bovine, murine (e.g., Mav1, described by Beard et al., Virol,75-81, 1990), ovine, porcine, avian, and simian (e.g., SAV) origin.Preferably, the adenovirus of animal origin is a canine adenovirus, morepreferably a CAV2 adenovirus (e.g., Manhattan or A26/61 strain ATCCVR-800, for example).

In another embodiment the gene can be introduced in a retroviral vector(e.g., as described in U.S. Pat. Nos. 5,399,346, 4,650,764, 4,980,289and 5,124,263; all of which are herein incorporated by reference; Mannet al., Cell, 33:153, 1983; Markowitz et al., J Virol, 62:1120, 1988;PCT/US95/14575; EP 453242; EP178220; Bernstein et al., Genet Eng, 7:235,1985; McCormick, BioTechnol, 3:689, 1985; International PatentPublication No. WO95/07358; and Kuo et al., Blood, 82:845, 1993). Theretroviruses are integrating viruses that infect dividing cells. Theretrovirus genome includes two LTRs, an encapsidation sequence and threecoding regions (gag, pol and env). In recombinant retroviral vectors,the gag, pol and env genes are generally deleted, in whole or in part,and replaced with a heterologous nucleic acid sequence of interest.These vectors can be constructed from different types of retrovirus,such as, HIV, MoMuLV (“murine Moloney leukaemia virus” MSV (“murineMoloney sarcoma virus”), HaSV (“Harvey sarcoma virus”); SNV (“spleennecrosis virus”); RSV (“Rous sarcoma virus”) and Friend virus. Defectiveretroviral vectors are disclosed in WO95/02697.

In general, in order to construct recombinant retroviruses containing anucleic acid sequence, a plasmid containing the LTRs, the encapsidationsequence and the coding sequence is constructed. This construct is usedto transfect a packaging cell line, which cell line is able to supply intrans the retroviral functions that are deficient in the plasmid. Ingeneral, the packaging cell lines are thus able to express the gag, poland env genes. Such packaging cell lines have been described in theprior art, in particular the cell line PA317 (U.S. Pat. No. 4,861,719,herein incorporated by reference); the PsiCRIP cell line (See,WO90/02806), and the GP+envAm-12 cell line (See, WO89/07150). Inaddition, the recombinant retroviral vectors can contain modificationswithin the LTRs for suppressing transcriptional activity as well asextensive encapsidation sequences which may include a part of the gaggene (Bender et al., J Virol, 61:1639, 1987). Recombinant retroviralvectors are purified by standard techniques known to those havingordinary skill in the art.

Alternatively, the vector can be introduced in vivo by lipofection. Forthe past decade, there has been increasing use of liposomes forencapsulation and transfection of nucleic acids in vitro. Syntheticcationic lipids designed to limit the difficulties and dangersencountered with liposome-mediated transfection can be used to prepareliposomes for in vivo transfection of a gene encoding a marker (Felgneret al., Proc Natl Acad Sci USA, 84:7413-7417, 1987; Mackey, et al., ProcNatl Acad Sci USA, 85:8027-8031, 1988; and Ulmer et al., Science,259:1745-1748, 1993). The use of cationic lipids may promoteencapsulation of negatively charged nucleic acids, and also promotefusion with negatively charged cell membranes (Felgner and Ringold,Science, 337:387-388, 1989). Particularly useful lipid compounds andcompositions for transfer of nucleic acids are described inInternational Patent Publications WO95/18863 and WO96/17823, and in U.S.Pat. No. 5,459,127, herein incorporated by reference.

Other molecules are also useful for facilitating transfection of anucleic acid in vivo, such as a cationic oligopeptide (e.g.,International Patent Publication WO95/21931), peptides derived from DNAbinding proteins (e.g., International Patent Publication WO96/25508), ora cationic polymer (e.g., International Patent Publication WO95/21931).

It is also possible to introduce the vector in vivo as a naked DNAplasmid. Methods for formulating and administering naked DNA tomammalian muscle tissue are disclosed in U.S. Pat. Nos. 5,580,859 and5,589,466, both of which are herein incorporated by reference.

DNA vectors for gene therapy can be introduced into the desired hostcells by methods known in the art, including but not limited totransfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a genegun, or use of a DNA vector transporter (See e.g., Wu et al., J BiolChem, 267:963-967, 1992; Wu and Wu, J Biol Chem, 263:14621-14624, 1988;and Williams et al., Proc Natl Acad Sci USA, 88:2726-2730, 1991).Receptor-mediated DNA delivery approaches can also be used (Curiel etal., Hum Gene Ther, 3:147-154, 1992; Wu and Wu, J Biol Chem,262:4429-4432, 1987).

The present invention also provides methods and compositions for theproduction of in vitro cell cultures that express wild-type or mutatedhuman α7, as well as transgenic animals capable of expressing wild-typeor mutated human α7. For example, the genomic α7 clone can be expressedin mammalian cells (e.g., cell lines, including but not limited tomammalian kidney cells, such as HEK). It is also contemplated that insome embodiments, the cells and animals also express other foreign genesin conjunction with the introduced α7.

The present invention also provides methods for producing non-humantransgenic animals, comprising the steps of: a) introducing into anembryonal cell of a non-human animal a polynucleotide sequence encodingan α7 protein; b) transplanting the transgenic embryonal target cellformed thereby into a recipient female parent; and c) identifying atleast one offspring containing the transgene wherein the α7 mRNA isoverexpressed in the tissue of the offspring. In one preferredembodiment, the α7 mRNA is human α7 mRNA. In an alternative embodiment,the polynucleotide sequence encoding an α7 protein comprises a yeastartificial chromosome, while in another embodiment, the polynucleotidesequence encoding an α7 is a bacterial artificial chromosome, and in yetanother embodiment, the polynucleotide sequence encoding an α7 proteinis a P1 artificial chromosome. In a further embodiment, the non-humananimal is a member of the Order Rodentia. In a preferred embodiment, thenon-human animal is a mouse.

DEFINITIONS

To facilitate understanding of the invention, a number of terms aredefined below.

The term “α7 gene” (or Alpha-7, or “Alpha-7 gene”) refers to thefull-length α7 nucleotide sequence. However, it is also intended thatthe term encompass fragments of the α7 sequence, such as those set forthas SEQ ID NOS:94, 101, 122, and 125, as well as other domains within thefull-length α7 nucleotide sequence. Furthermore, the terms “Alpha-7nucleotide sequence” or “Alpha-7 polynucleotide sequence” (or “α7nucleotide sequence” or “α7 polynucleotide sequence”) encompasses DNA,cDNA, and RNA (e.g., mRNA) sequences. In preferred embodiments, the α7is human α7.

A “variant” of human α7 as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties, (e.g., replacement of leucine withisoleucine). More rarely, a variant may have “nonconservative” changes(e.g., replacement of a glycine with a tryptophan). Similar minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

The term “biologically active,” as used herein, refers to a protein orother biologically active molecules (e.g., catalytic RNA) havingstructural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, “immunologically active” refers to thecapability of the natural, recombinant, or synthetic α7, or anyoligopeptide or polynucleotide thereof, to induce a specific immuneresponse in appropriate animals or cells and to bind with specificantibodies.

The term “agonist,” as used herein, refers to a molecule which, whenbound to α7, causes a change in α7, which modulates the activity of α7.Agonists may include proteins, nucleic acids, carbohydrates, or anyother molecules that bind to or interact with α7.

The terms “antagonist” or “inhibitor,” as used herein, refer to amolecule which, when bound to α7, blocks or modulates the biological orimmunological activity of α7. Antagonists and inhibitors may includeproteins, nucleic acids, carbohydrates, or any other molecules, whichbind or interact with α7.

The term “modulate,” as used herein, refers to a change or an alterationin the biological activity of α7. Modulation may be an increase or adecrease in protein activity, a change in binding characteristics, orany other change in the biological, functional, or immunologicalproperties of α7.

The term “derivative,” as used herein, refers to the chemicalmodification of a nucleic acid encoding α7, or the encoded α7.Illustrative of such modifications would be replacement of hydrogen byan alkyl, acyl, or amino group. A nucleic acid derivative would encode apolypeptide that retains essential biological characteristics of thenatural molecule.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of a polypeptideor precursor (e.g., human α7). The polypeptide can be encoded by a fulllength coding sequence or by any portion of the coding sequence so longas the desired activity or functional properties (e.g., enzymaticactivity, ligand binding, signal transduction, etc.) of the full-lengthor fragment are retained. The term also encompasses the coding region ofa structural gene and the including sequences located adjacent to thecoding region on both the 5′ and 3′ ends for a distance of about 1 kb oneither end such that the gene corresponds to the length of thefull-length mRNA. The sequences which are located 5′ of the codingregion and which are present on the mRNA are referred to as 5′non-translated sequences. The sequences which are located 3′ ordownstream of the coding region and which are present on the mRNA arereferred to as 3′ non-translated sequences. The term “gene” encompassesboth cDNA and genomic forms of a gene.

A genomic form or clone of a gene contains the coding region interruptedwith non-coding sequences termed “introns” or “intervening regions” or“intervening sequences.” Introns are segments of a gene that aretranscribed into nuclear RNA (hnRNA). Introns may contain regulatoryelements such as enhancers. Introns are removed or “spliced out” fromthe nuclear or primary transcript, and thus introns are absent in themessenger RNA (mRNA) transcript. The mRNA functions during translationto specify the sequence or order of amino acids in a nascentpolypeptide.

Where “amino acid sequence” is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, “amino acidsequence” and like terms, such as “polypeptide” or “protein,” are notmeant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

In addition to containing introns, genomic forms of a gene may alsoinclude sequences located on both the 5′ and 3′ end of the sequencesthat are present on the RNA transcript. These sequences are referred toas “flanking” sequences or regions (these flanking sequences are located5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers that control or influence thetranscription of the gene. The 3′ flanking region may contain sequencesthat direct the termination of transcription, post-transcriptionalcleavage and polyadenylation. The present invention provides DNAsequence of the α7 promoter (SEQ ID NO:101; See, FIG. 8). The presentinvention also provides DNA sequence for the region located 5′ to thehuman α7 gene (SEQ ID NO:94; See, FIG. 4).

“Peptide nucleic acid,” as used herein, refers to a molecule thatcomprises an oligomer to which an amino acid residue, such as lysine,and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary strand of nucleic acid (Nielsen et al., AnticancerDrug Des, 8:53-63, 1993).

The term “wild-type” refers to a gene or gene product that has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene. In contrast, the term“modified” or “mutant” refers to a gene or gene product that displaysmodifications in sequence and or functional properties (i.e., alteredcharacteristics) when compared to the wild-type gene or gene product. Itis noted that naturally occurring mutants can be isolated; these areidentified by the fact that they have altered characteristics whencompared to the wild-type gene or gene product.

As used herein, the term “polymorphism” refers to the regular andsimultaneous occurrence in a single interbreeding population of two ormore alleles of a gene, where the frequency of the rarer allele(s) isgreater than can be explained by recurrent mutation alone (typicallygreater than 1%). In preferred embodiments, the term “polymorphism”refers to at least one substitution, insertion and/or deletion in the 5′untranslated region of α7. In particularly preferred embodiments, thepolymorphism is in the α7 promoter and contributes to a reduction in α7transcription. In other preferred embodiments, the polymorphism isassociated with a predisposition to schizophrenia.

The term “allele” refers to one of at least two mutually exclusive formsof the same gene, occupying the same locus on homologous chromosomes,and governing the same biochemical and developmental process.

As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

DNA molecules are said to have “5′ends” and “3′ends” becausemononucleotides are reacted to make oligonucleotides or polynucleotidesin a manner such that the 5′ phosphate of one mononucleotide pentosering is attached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage. Therefore, an end of an oligonucleotide or apolynucleotide, referred to as the “5′end” if its 5′ phosphate is notlinked to the 3′ oxygen of a mononucleotide pentose ring and as the“3′end” if its 3′ oxygen is not linked to a 5′ phosphate of a subsequentmononucleotide pentose ring. As used herein, a nucleic acid sequence,even if internal to a larger oligonucleotide or polynucleotide, also maybe said to have 5′ and 3′ ends. In either a linear or circular DNAmolecule, discrete elements are referred to as being “upstream” or 5′ ofthe “downstream” or 3′ elements. This terminology reflects the fact thattranscription proceeds in a 5′ to 3′ fashion along the DNA strand. Thepromoter and enhancer elements which direct transcription of a linkedgene are generally located 5′ or upstream of the coding region. However,enhancer elements can exert their effect even when located 3′ of thepromoter element and the coding region. Transcription termination andpolyadenylation signals are located 3′ or downstream of the codingregion.

As used herein, the terms “an oligonucleotide having a nucleotidesequence encoding a gene” and “polynucleotide having a nucleotidesequence encoding a gene,” means a nucleic acid sequence comprising thecoding region of a gene or in other words the nucleic acid sequence thatencodes a gene product. The coding region may be present in either acDNA, a genomic DNA or an RNA form. When present in a DNA form, theoligonucleotide or polynucleotide may be single-stranded (i.e., thesense strand) or double-stranded. Suitable control elements such asenhancers/promoters, splice junctions, polyadenylation signals, etc. maybe placed in close proximity to the coding region of the gene if neededto permit proper initiation of transcription and/or correct processingof the primary RNA transcript. Alternatively, the coding region utilizedin the expression vectors of the present invention may containendogenous enhancers/promoters, splice junctions, intervening sequences,polyadenylation signals, etc. or a combination of both endogenous andexogenous control elements.

As used herein, the term “regulatory element” refers to a geneticelement that controls some aspect of the expression of nucleic acidsequences. For example, a promoter is a regulatory element thatfacilitates the initiation of transcription of an operably linked codingregion. Other regulatory elements are splicing signals, polyadenylationsignals, termination signals, etcetera.

Transcriptional control signals in eukaryotes comprise “promoter” and“enhancer” elements, and in some cases further comprise operatorsequences. Promoters and enhancers consist of short arrays of DNAsequences that interact specifically with cellular proteins involved intranscription (Maniatis et al., Science, 236:1237, 1987). Promoter andenhancer elements have been isolated from a variety of eukaryoticsources including genes in yeast, insect and mammalian cells and viruses(analogous control elements, i.e., promoters, are also found inprokaryote). The selection of a particular promoter and enhancer dependson what cell type is to be used to express the protein of interest. Someeukaryotic promoters and enhancers have a broad host range while othersare functional in a limited subset of cell types (Voss et al., TrendsBiochem Sci, 11:287, 1986; and Maniatis et al., supra, 1987). Forexample, the SV40 early gene enhancer is very active in a wide varietyof cell types from many mammalian species and has been widely used forthe expression of proteins in mammalian cells (Dijkema et al., EMBO J,4:761, 1985). Two other examples of promoter/enhancer elements active ina broad range of mammalian cell types are those from the humanelongation factor 1α gene (Uetsuki et al., J Biol Chem, 264:5791, 1989;Kim et al., Gene 91:217, 1990; and Mizushima and Nagata, Nuc Acids Res,18:5322, 1990) and the long terminal repeats of the Rous sarcoma virus(Gorman et al., Proc Natl Acad Sci USA, 79:6777, 1982) and the humancytomegalovirus (Boshart et al., Cell, 41:521, 1985).

As used herein, the term “promoter/enhancer” denotes a segment of DNAwhich contains sequences capable of providing both promoter and enhancerfunctions (i.e., the functions provided by a promoter element and anenhancer element, see above for a discussion of these functions). Forexample, the long terminal repeats of retroviruses contain both promoterand enhancer functions. The enhancer/promoter may be “endogenous” or“exogenous” or “heterologous.” An “endogenous” enhancer/promoter is onethat is naturally linked with a given gene in the genome. An “exogenous”or “heterologous” enhancer/promoter is one that is placed injuxtaposition to a gene by means of genetic manipulation (i.e.,molecular biological techniques) such that transcription of that gene isdirected by the linked enhancer/promoter.

The term “repressor” as used herein refers to a regulatory protein thatbinds to an operator of a gene to prevent transcription of the gene. Thebinding affinity of repressors for the operator may be affected by othermolecules. Inducers bind to repressors and decrease their binding to theoperator, while corepressors increase the binding. As used herein, theterms “operator” and “repressor sequence” refer to the site on DNA towhich a specific repressor protein binds thereby preventing theinitiation of transcription at the adjacent promoter.

The presence of “splicing signals” on an expression vector often resultsin higher levels of expression of the recombinant transcript. Splicingsignals mediate the removal of introns from the primary RNA transcriptand consist of a splice donor and acceptor site (Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, New York, pp. 16.6-16.8, 1989). A commonly used splicedonor and acceptor site is the splice junction from the 16S RNA of SV40.

Efficient expression of recombinant DNA sequences in eukaryotic cellsrequires expression of signals directing the efficient termination andpolyadenylation of the resulting transcript. Transcription terminationsignals are generally found downstream of the polyadenylation signal andare a few hundred nucleotides in length. The term “polyA site” or “polyAsequence” as used herein denotes a DNA sequence, which directs both thetermination and polyadenylation of the nascent RNA transcript. Efficientpolyadenylation of the recombinant transcript is desirable astranscripts lacking a polyA tail are unstable and are rapidly degraded.The polyA signal utilized in an expression vector may be “heterologous”or “endogenous.” An endogenous polyA signal is one that is foundnaturally at the 3′ end of the coding region of a given gene in thegenome. A heterologous polyA signal is one that is isolated from onegene and placed 3′ of another gene. A commonly used heterologous polyAsignal is the SV40 polyA signal. The SV40 polyA signal is contained on a237 bp BamHI/BclI restriction fragment and directs both termination andpolyadenylation (Sambrook, supra, 1989).

Eukaryotic expression vectors may also contain “viral replicons” or“viral origins of replication.” Viral replicons are viral DNA sequencesthat allow for the extrachromosomal replication of a vector in a hostcell expressing the appropriate replication factors. Vectors thatcontain either the SV40 or polyoma virus origin of replication replicateto high copy number (up to 104 copies/cell) in cells that express theappropriate viral T antigen. Vectors that contain the replicons frombovine papillomavirus or Epstein-Barr virus replicate extrachromosomallyat low copy number (˜100 copies/cell).

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, for the sequence“A-G-T,” is complementary to the sequence “T-C-A.” Complementarity maybe “partial,” in which only some of the nucleic acids' bases are matchedaccording to the base pairing rules. Or, there may be “complete” or“total” complementarity between the nucleic acids. The degree ofcomplementarity between nucleic acid strands has significant effects onthe efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions, aswell as detection methods that depend upon binding between nucleicacids.

The term “homology” refers to a degree of complementarity. There may bepartial homology or complete homology (i.e., identity). A partiallycomplementary sequence is one that at least partially inhibits acompletely complementary sequence from hybridizing to a target nucleicacid is referred to using the functional term “substantiallyhomologous.” The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (Southern or Northern blot, solution hybridizationand the like) under conditions of low stringency. A substantiallyhomologous sequence or probe will compete for and inhibit the binding(i.e., the hybridization) of a completely homologous to a target underconditions of low stringency. This is not to say that conditions of lowstringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second target whichlacks even a partial degree of complementarity (e.g., less than about30% identity); in the absence of non-specific binding the probe will nothybridize to the second non-complementary target.

The art knows well that numerous equivalent conditions may be employedto comprise low stringency conditions; factors such as the length andnature (DNA, RNA, base composition) of the probe and nature of thetarget (DNA, RNA, base composition, present in solution or immobilized,etc.) and the concentration of the salts and other components (e.g., thepresence or absence of formamide, dextran sulfate, polyethylene glycol)are considered and the hybridization solution may be varied to generateconditions of low stringency hybridization different from, butequivalent to, the above listed conditions. In addition, the art knowsconditions that promote hybridization under conditions of highstringency (e.g., increasing the temperature of the hybridization and/orwash steps, the use of formamide in the hybridization solution, etc.).

When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or genomic clone, the term “substantially homologous” refersto any probe which can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low stringencyas described above.

A gene may produce multiple RNA species, which are generated bydifferential splicing of the primary RNA transcript. cDNAs that aresplice variants of the same gene will contain regions of sequenceidentity or complete homology (representing the presence of the sameexon or portion of the same exon on both cDNAs) and regions of completenon-identity (for example, representing the presence of exon “A” on cDNA1 wherein cDNA 2 contains exon “B” instead). Because the two cDNAscontain regions of sequence identity they will both hybridize to a probederived from the entire gene or portions of the gene containingsequences found on both cDNAs; the two splice variants are thereforesubstantially homologous to such a probe and to each other.

When used in reference to a single-stranded nucleic acid sequence, theterm “substantially homologous” refers to any probe which can hybridize(i.e., it is the complement of) the single-stranded nucleic acidsequence under conditions of low stringency as described above.

The term “hybridization” is used in reference to the pairing ofcomplementary nucleic acids. Hybridization and the strength ofhybridization (i.e., the strength of the association between the nucleicacids) is impacted by such factors as the degree of complementarybetween the nucleic acids, stringency of the conditions involved, theT_(m) of the formed hybrid, and the G:C ratio within the nucleic acids.A single molecule that contains pairing of complementary nucleic acidswithin its structure is said to be “self-hybridized.”

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. The equation for calculating the T_(m)of nucleic acids is well known in the art. As indicated by standardreferences, a simple estimate of the T_(m) value may be calculated bythe equation: T_(m)=81.5+0.41(% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl (See e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization, 1985). Other referencesinclude more sophisticated computations that take structural as well assequence characteristics into account for the calculation of T_(m).

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. Under “low stringency conditions” anucleic acid sequence of interest will hybridize to its exactcomplement, sequences with single base mismatches, closely relatedsequences (e.g., sequences with 90% or greater homology), and sequenceshaving only partial homology (e.g., sequences with 50-90% homology).Under ‘medium stringency conditions,” a nucleic acid sequence ofinterest will hybridize only to its exact complement, sequences withsingle base mismatches, and closely relation sequences (e.g., 90% orgreater homology). Under “high stringency conditions,” a nucleic acidsequence of interest will hybridize only to its exact complement, and(depending on conditions such a temperature) sequences with single basemismatches. In other words, under conditions of high stringency thetemperature can be raised so as to exclude hybridization to sequenceswith single base mismatches.

“High stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 0.1×SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

“Medium stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 1.0×SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

“Low stringency conditions” comprise conditions equivalent to binding orhybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.1% SDS, 5×Denhardt's reagent [50×Denhardt's contains per 500ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and100 μg/ml denatured salmon sperm DNA followed by washing in a solutioncomprising 5×SSPE, 0.1% SDS at 42° C. when a probe of about 500nucleotides in length is employed.

The art knows well that numerous equivalent conditions may be employedto comprise low stringency conditions; factors such as the length andnature (DNA, RNA, base composition) of the probe and nature of thetarget (DNA, RNA, base composition, present in solution or immobilized,etc.) and the concentration of the salts and other components (e.g., thepresence or absence of formamide, dextran sulfate, polyethylene glycol)are considered and the hybridization solution may be varied to generateconditions of low stringency hybridization different from, butequivalent to, the above listed conditions. In addition, the art knowsconditions that promote hybridization under conditions of highstringency (e.g., increasing the temperature of the hybridization and/orwash steps, the use of formamide in the hybridization solution, etc.)(see definition above for “stringency”).

“Amplification” is a special case of nucleic acid replication involvingtemplate specificity. It is to be contrasted with non-specific templatereplication (i.e., replication that is template-dependent but notdependent on a specific template). Template specificity is heredistinguished from fidelity of replication (i.e., synthesis of theproper polynucleotide sequence) and nucleotide (ribo- ordeoxyribo-)specificity. Template specificity is frequently described interms of “target” specificity. Target sequences are “targets” in thesense that they are sought to be sorted out from other nucleic acid.Amplification techniques have been designed primarily for this sortingout.

Template specificity is achieved in most amplification techniques by thechoice of enzyme. Amplification enzymes are enzymes that, underconditions they are used, will process only specific sequences ofnucleic acid in a heterogeneous mixture of nucleic acid. For example, inthe case of Qβ replicase, MDV-1 RNA is the specific template for thereplicase (Kacian et al., Proc Natl Acad Sci USA, 69:3038, 1972). Thisamplification enzyme does not replicate other nucleic acids. Similarly,in the case of T7 RNA polymerase, this amplification enzyme has astringent specificity for its own promoters (Chamberlin et al., Nature,228:227, 1970). In the case of T4 DNA ligase, the enzyme will not ligatethe two oligonucleotides or polynucleotides, where there is a mismatchbetween the oligonucleotide or polynucleotide substrate and the templateat the ligation junction (Wu and Wallace, Genomics, 4:560, 1989).Finally, Taq and Pfu polymerases, by virtue of their ability to functionat high temperature, are found to display high specificity for thesequences bounded and thus defined by the primers; the high temperatureresults in thermodynamic conditions that favor primer hybridization withthe target sequences and not hybridization with non-target sequences(Erlich (ed.), PCR Technology, Stockton Press, 1989).

As used herein, the term “amplifiable nucleic acid” is used in referenceto nucleic acids that may be amplified by any amplification method. Itis contemplated that “amplifiable nucleic acid” will usually comprise“sample template.”

As used herein, the term “sample template” refers to nucleic acidoriginating from a sample, which is analyzed for the presence of“target” (defined below). In contrast, “background template” is used inreference to nucleic acid other than sample template, which may or maynot be present in a sample. Background template is most ofteninadvertent. It may be the result of carryover, or it may be due to thepresence of nucleic acid contaminants sought to be purified away fromthe sample. For example, nucleic acids from organisms other than thoseto be detected may be present as background in a test sample.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method. Thepresent invention provides sequences for numerous primers (i.e., SEQ IDNOS:1-8, and 12-83).

The term “sense primer” refers to an oligonucleotide capable ofhybridizing to the noncoding strand of gene. The term “antisense primer”refers to an oligonucleotide capable of hybridizing to the coding strandof a gene.

As used herein, the term “fluorescent tag” refers to a molecule havingthe ability to emit light of a certain wavelength when activated bylight of another wavelength. “Fluorescent tags” suitable for use withthe present invention include but are not limited to fluorescein,rhodamine, Texas red, 6-FAM, TET, HEX, Cy5, Cy3, and Oregon Green.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, which is capable of hybridizing to anotheroligonucleotide of interest. A probe may be single-stranded ordouble-stranded. Probes are useful in the detection, identification andisolation of particular gene sequences. It is contemplated that anyprobe used in the present invention will be labeled with any “reportermolecule,” so that is detectable in any detection system, including, butnot limited to enzyme (e.g., ELISA, as well as enzyme-basedhistochemical assays), fluorescent, radioactive, and luminescentsystems. It is not intended that the present invention be limited to anyparticular detection system or label. The present invention providessequences for suitable for use as probes (e.g., SEQ ID NO:9-11, as wellas the primer sequences described above).

As used herein, the term “target,” when used in reference to thepolymerase chain reaction, refers to the region of nucleic acid boundedby the primers used for polymerase chain reaction. Thus, the “target” issought to be sorted out from other nucleic acid sequences. A “segment”is defined as a region of nucleic acid within the target sequence.

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe method of Mullis (See e.g., U.S. Pat. Nos. 4,683,195 4,683,202, and4,965,188, hereby incorporated by reference), which describe a methodfor increasing the concentration of a segment of a target sequence in amixture of genomic DNA without cloning or purification. This process foramplifying the target sequence consists of introducing a large excess oftwo oligonucleotide primers to the DNA mixture containing the desiredtarget sequence, followed by a precise sequence of thermal cycling inthe presence of a DNA polymerase. The two primers are complementary totheir respective strands of the double stranded target sequence. Toeffect amplification, the mixture is denatured and the primers thenannealed to their complementary sequences within the target molecule.Following annealing, the primers are extended with a polymerase so as toform a new pair of complementary strands. The steps of denaturation,primer annealing and polymerase extension can be repeated many times(i.e., denaturation, annealing and extension constitute one “cycle”;there can be numerous “cycles”) to obtain a high concentration of anamplified segment of the desired target sequence. The length of theamplified segment of the desired target sequence is determined by therelative positions of the primers with respect to each other, andtherefore, this length is a controllable parameter. By virtue of therepeating aspect of the process, the method is referred to as the“polymerase chain reaction” (hereinafter “PCR”). Because the desiredamplified segments of the target sequence become the predominantsequences (in terms of concentration) in the mixture, they are said tobe “PCR amplified”.

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level detectable by several differentmethodologies (e.g., hybridization with a labeled probe; incorporationof biotinylated primers followed by avidin-enzyme conjugate detection;incorporation of ³²P-labeled deoxynucleotide triphosphates, such as dCTPor dATP, into the amplified segment). In addition to genomic DNA, anyoligonucleotide or polynucleotide sequence can be amplified with theappropriate set of primer molecules. In particular, the amplifiedsegments created by the PCR process itself are, themselves, efficienttemplates for subsequent PCR amplifications.

As used herein, the terms “PCR product,” “PCR fragment,” and“amplification product” refer to the resultant mixture of compoundsafter two or more cycles of the PCR steps of denaturation, annealing andextension are complete. These terms encompass the case where there hasbeen amplification of one or more segments of one or more targetsequences.

As used herein, the term “amplification reagents” refers to thosereagents (deoxyribonucleotide triphosphates, buffer, etc.), needed foramplification except for primers, nucleic acid template and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.).

As used herein, the term “RT-PCR” refers to the replication andamplification of RNA sequences. In this method, reverse transcription iscoupled to PCR, most often using a single enzyme procedure in which athermostable polymerase is employed, as described in U.S. Pat. No.5,322,770, herein incorporated by reference. In RT-PCR, the RNA templateis converted to cDNA due to the reverse transcriptase activity of thepolymerase, and then amplified using the polymerizing activity of thepolymerase (i.e., as in other PCR methods).

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

As used herein, the term “recombinant DNA molecule” as used hereinrefers to a DNA molecule comprising segments of DNA joined together bymeans of molecular biological techniques.

As used herein, the term “antisense” is used in reference to RNAsequences that are complementary to a specific RNA sequence (e.g.,mRNA). Included within this definition are antisense RNA (“asRNA”)molecules involved in gene regulation by bacteria. Antisense RNA may beproduced by any method, including synthesis by splicing the gene(s) ofinterest in a reverse orientation to a viral promoter, permitting thesynthesis of a coding strand. Once introduced into an embryo, thistranscribed strand combines with natural mRNA produced by the embryo toform duplexes. These duplexes then block either the furthertranscription of the mRNA or its translation. In this manner, mutantphenotypes may be generated. The term “antisense strand” is used inreference to a nucleic acid strand that is complementary to the “sense”strand. The designation (−) (i.e., “negative”) is sometimes used inreference to the antisense strand, with the designation (+) sometimesused in reference to the sense (i.e., “positive”) strand.

As used herein, the term “polyA⁺ RNA” refers to RNA molecules having astretch of adenine nucleotides at the 3′ end. This polyadenine stretchis also referred to as a “poly-A tail.” Eukaryotic mRNA moleculescontain poly-A tails and are referred to as polyA⁺ RNA.

The terms “in operable combination,” “in operable order,” and “operablylinked” as used herein refer to the linkage of nucleic acid sequences insuch a manner that a nucleic acid molecule capable of directing thetranscription of a given gene and/or the synthesis of a desired proteinmolecule is produced. The term also refers to the linkage of amino acidsequences in such a manner so that a functional protein is produced.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” or “isolated polynucleotide” refers to anucleic acid sequence that is identified and separated from at least onecontaminant nucleic acid with which it is ordinarily associated in itsnatural source. Isolated nucleic acid is such present in a form orsetting that is different from that in which it is found in nature. Incontrast, non-isolated nucleic acids as nucleic acids such as DNA andRNA found in the state they exist in nature. For example, a given DNAsequence (e.g., a gene) is found on the host cell chromosome inproximity to neighboring genes; RNA sequences, such as a specific mRNAsequence encoding a specific protein, are found in the cell as a mixturewith numerous other mRNAs, which encode a multitude of proteins.However, isolated nucleic acid encoding a mammalian α7 protein includes,by way of example, such nucleic acid in cells ordinarily expressing anα7 protein where the nucleic acid is in a chromosomal location differentfrom that of natural cells, or is otherwise flanked by a differentnucleic acid sequence than that found in nature. The isolated nucleicacid, oligonucleotide, or polynucleotide may be present insingle-stranded or double-stranded form. When an isolated nucleic acid,oligonucleotide or polynucleotide is to be utilized to express aprotein, the oligonucleotide or polynucleotide will contain at a minimumthe sense or coding strand (i.e., the oligonucleotide or polynucleotidemay single-stranded), but may contain both the sense and anti-sensestrands (i.e., the oligonucleotide or polynucleotide may bedouble-stranded).

As used herein, a “portion of a chromosome” refers to a discrete sectionof the chromosome. Chromosomes are divided into sites or sections bycytogeneticists as follows: the short (relative to the centromere) armof a chromosome is termed the “p” arm; the long arm is termed the “q”arm. Each arm is then divided into 2 regions termed region 1 and region2 (region 1 is closest to the centromere). Each region is furtherdivided into bands. The bands may be further divided into sub-bands. Forexample, the 11p15.5 portion of human chromosome 11 is the portionlocated on chromosome 11 (11) on the short arm (p) in the first region(1) in the 5th band (5) in sub-band 5 (0.5). A portion of a chromosomemay be “altered;” for instance the entire portion may be absent due to adeletion or may be rearranged (e.g., inversions, translocations,expanded or contracted due to changes in repeat regions). In the case ofa deletion, an attempt to hybridize (i.e., specifically bind) a probehomologous to a particular portion of a chromosome could result in anegative result (i.e., the probe could not bind to the sample containinggenetic material suspected of containing the missing portion of thechromosome). Thus, hybridization of a probe homologous to a particularportion of a chromosome may be used to detect alterations in a portionof a chromosome.

The term “sequences associated with a chromosome” means preparations ofchromosomes (e.g., spreads of metaphase chromosomes), nucleic acidextracted from a sample containing chromosomal DNA (e.g., preparationsof genomic DNA); the RNA which is produced by transcription of geneslocated on a chromosome (e.g., hnRNA and mRNA) and cDNA copies of theRNA transcribed from the DNA located on a chromosome. Sequencesassociated with a chromosome may be detected by numerous techniquesincluding probing of Southern and Northern blots and in situhybridization to RNA, DNA or metaphase chromosomes with probescontaining sequences homologous to the nucleic acids in the above listedpreparations.

As used herein the term “coding region” when used in reference tostructural gene refers to the nucleotide sequences which encode theamino acids found in the nascent polypeptide as a result of translationof a mRNA molecule. The coding region is bounded, in eukaryotes, on the5′ side by the nucleotide triplet “ATG” which encodes the initiatormethionine and on the 3′ side by one of the three triplets that specifystop codons (i.e., TAA, TAG, TGA).

As used herein, the term “structural gene” refers to a DNA sequencecoding for RNA or a protein. In contrast, “regulatory genes” arestructural genes that encode products that control the expression ofother genes (e.g., transcription factors).

As used herein, the term “purified” or “to purify” refers to the removalof contaminants from a sample. For example, anti-α7 antibodies arepurified by removal of contaminating non-immunoglobulin proteins; theyare also purified by the removal of immunoglobulin that does not bindα7. The removal of non-immunoglobulin proteins and/or the removal ofimmunoglobulins that do not bind α7, results in an increase in thepercent of α7-reactive immunoglobulins in the sample. In anotherexample, recombinant α7 polypeptides are expressed in bacterial hostcells and the polypeptides are purified by the removal of host cellproteins; the percent of recombinant α7 polypeptides is therebyincreased in the sample.

The term “recombinant DNA molecule” as used herein refers to a DNAmolecule that is comprised of segments of DNA joined together by meansof molecular biological techniques.

The term “recombinant protein” or “recombinant polypeptide” as usedherein refers to a protein molecule that is expressed from a recombinantDNA molecule.

The term “native protein” as used herein to indicate that a protein doesnot contain amino acid residues encoded by vector sequences; that is thenative protein contains only those amino acids found in the protein asit occurs in nature. A native protein may be produced by recombinantmeans or may be isolated from a naturally occurring source.

As used herein the term “portion” when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid.

The term “portion” when used in reference to a nucleotide sequencerefers to fragments of that nucleotide sequence. The fragments may rangein size from 5 nucleotide residues to the entire nucleotide sequenceminus one nucleic acid residue.

As used herein, the term “fusion protein” refers to a chimeric proteincontaining the protein of interest (i.e., mouse or human α7 andfragments thereof) joined to an exogenous protein fragment (the fusionpartner which consists of a non-α7 protein). The fusion partner mayenhance solubility of the α7 protein as expressed in a host cell, mayprovide an affinity tag to allow purification of the recombinant fusionprotein from the host cell or culture supernatant, or both. If desired,the fusion protein may be removed from the protein of interest (i.e., α7protein or fragments thereof) by a variety of enzymatic or chemicalmeans known to the art.

The term “Southern blot,” refers to the analysis of DNA on agarose oracrylamide gels to fractionate the DNA according to size followed bytransfer of the DNA from the gel to a solid support, such asnitrocellulose or a nylon membrane. The immobilized DNA is then probedwith a labeled probe to detect DNA species complementary to the probeused. The DNA may be cleaved with restriction enzymes prior toelectrophoresis. Following electrophoresis, the DNA may be partiallydepurinated and denatured prior to or during transfer to the solidsupport. Southern blots are a standard tool of molecular biologists(Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Press, NY, pp 9.31-9.58, 1989).

The term “Northern blot,” as used herein refers to the analysis of RNAby electrophoresis of RNA on agarose gels to fractionate the RNAaccording to size followed by transfer of the RNA from the gel to asolid support, such as nitrocellulose or a nylon membrane. Theimmobilized RNA is then probed with a labeled probe to detect RNAspecies complementary to the probe used. Northern blots are a standardtool of molecular biologists (Sambrook et al., supra, pp 7.39-7.52,1989).

The term “Western blot” refers to the analysis of protein(s) (orpolypeptides) immobilized onto a support such as nitrocellulose or amembrane. The proteins are run on acrylamide gels to separate theproteins, followed by transfer of the protein from the gel to a solidsupport, such as nitrocellulose or a nylon membrane. The immobilizedproteins are then exposed to antibodies with reactivity against anantigen of interest. The binding of the antibodies may be detected byvarious methods, including the use of radiolabeled antibodies.

The term “antigenic determinant” as used herein refers to that portionof an antigen that makes contact with a particular antibody (i.e., anepitope). When a protein or fragment of a protein is used to immunize ahost animal, numerous regions of the protein may induce the productionof antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the “immunogen” used to elicitthe immune response) for binding to an antibody.

The terms “specific binding” or specifically binding” when used inreference to the interaction of an antibody and a protein or peptidemeans that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A,” the presence of aprotein containing epitope A (or free, unlabelled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody.

The present invention also contemplates “non-human animals” comprisingany non-human animal capable of overexpressing α7 mRNA and/or proteins.Such non-human animals include vertebrates such as rodents, non-humanprimates, ovines, bovines, ruminants, lagomorphs, porcines, caprines,equines, canines, felines, aves, etc. Preferred non-human animals areselected from the order Rodentia, most preferably mice. The term “orderRodentia” refers to rodents (i.e., placental mammals [Class Euthria]which include the family Muridae (rats and mice).

The “non-human animals having a genetically engineered genotype” of theinvention are preferably produced by experimental manipulation of thegenome of the germline of the non-human animal. These geneticallyengineered non-human animals may be produced by several methodsincluding the introduction of a “transgene” comprising nucleic acid(usually DNA) into an embryonal target cell or integration into achromosome of the somatic and/or germ line cells of a non-human animalby way of human intervention, such as by the methods described herein.Non-human animals that contain a transgene are referred to as“transgenic non-human animals.” A transgenic animal is an animal whosegenome has been altered by the introduction of a transgene.

The term “transgene” as used herein refers to a foreign gene that isplaced into an organism by introducing the foreign gene into newlyfertilized eggs or early embryos. The term “foreign gene” refers to anynucleic acid (e.g., gene sequence) that is introduced into the genome ofan animal by experimental manipulations and may include gene sequencesfound in that animal so long as the introduced gene does not reside inthe same location as does the naturally occurring gene.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another. Theterm “vehicle” is sometimes used interchangeably with “vector.”

The term “expression vector” as used herein refers to a recombinant DNAmolecule containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host organism. Nucleic acid sequencesnecessary for expression in prokaryotes usually include a promoter, anoperator (optional), and a ribosome-binding site, often along with othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals.

The terms “overexpression” and “overexpressing” and grammaticalequivalents, are used in reference to levels of mRNA to indicate a levelof expression approximately 3-fold higher than that typically observedin a given tissue in a control or non-transgenic animal. Levels of mRNAare measured using any of a number of techniques known to those skilledin the art including, but not limited to Northern blot analysis.Appropriate controls are included on the Northern blot to control fordifferences in the amount of RNA loaded from each tissue analyzed (e.g.,the amount of 28S rRNA, an abundant RNA transcript present atessentially the same amount in all tissues, present in each sample canbe used as a means of normalizing or standardizing the α7 mRNA-specificsignal observed on Northern blots). The amount of mRNA present in theband corresponding in size to the correctly spliced α7 transgene RNA isquantified; other minor species of RNA which hybridize to the transgeneprobe are not considered in the quantification of the expression of thetransgenic mRNA.

The term “transfection” as used herein refers to the introduction offoreign DNA into eukaryotic cells. Transfection may be accomplished by avariety of means known to the art including calcium phosphate-DNAco-precipitation, DEAE-dextran-mediated transfection, polybrene-mediatedtransfection, electroporation, microinjection, liposome fusion,lipofection, protoplast fusion, retroviral infection, and biolistics.

The term “stable transfection” or “stably transfected” refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell, whichhas stably integrated foreign DNA into the genomic DNA.

The term “transient transfection” or “transiently transfected” refers tothe introduction of foreign DNA into a cell where the foreign DNA failsto integrate into the genome of the transfected cell. The foreign DNApersists in the nucleus of the transfected cell for several days. Duringthis time the foreign DNA is subject to the regulatory controls thatgovern the expression of endogenous genes in the chromosomes. The term“transient transfectant” refers to cells that have taken up foreign DNAbut have failed to integrate this DNA.

The term “calcium phosphate co-precipitation” refers to a technique forthe introduction of nucleic acids into a cell. The uptake of nucleicacids by cells is enhanced when the nucleic acid is presented as acalcium phosphate-nucleic acid co-precipitate. The original technique ofGraham and van der Eb (Graham and van der Eb, Virol, 52:456, 1973), hasbeen modified by several groups to optimize conditions for particulartypes of cells. The art is well aware of these numerous modifications.

As used herein, the term “selectable marker” refers to the use of a genewhich encodes an enzymatic activity that confers the ability to grow inmedium lacking what would otherwise be an essential nutrient (e.g., theHIS3 gene in yeast cells); in addition, a selectable marker may conferresistance to an antibiotic or drug upon the cell in which theselectable marker is expressed. Selectable markers may be “dominant”; adominant selectable marker encodes an enzymatic activity, which can bedetected in any eukaryotic cell line. Examples of dominant selectablemarkers include the bacterial aminoglycoside 3′ phosphotransferase gene(also referred to as the neo gene) which confers resistance to the drugG418 in mammalian cells, the bacterial hygromycin G phosphotransferase(hyg) gene which confers resistance to the antibiotic hygromycin and thebacterial xanthine-guanine phosphoribosyl transferase gene (alsoreferred to as the gpt gene) which confers the ability to grow in thepresence of mycophenolic acid. Other selectable markers are not dominantin that there use must be in conjunction with a cell line that lacks therelevant enzyme activity. Examples of non-dominant selectable markersinclude the thymidine kinase (tk) gene, which is used in conjunctionwith tk⁻ cell lines, the CAD gene, which is used in conjunction withCAD-deficient cells and the mammalian hypoxanthine-guaninephosphoribosyl transferase (hprt) gene, which is used in conjunctionwith hprt⁻ cell lines. A review of the use of selectable markers inmammalian cell lines is provided in Sambrook et al., Molecular Cloning:A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, NewYork, pp. 16.9-16.15, 1989.

As used herein, the term “cell culture” refers to any in vitro cultureof cells. Included within this term are continuous cell lines (e.g.,with an immortal phenotype), primary cell cultures, finite cell lines(e.g., non-transformed cells), and any other cell population maintainedin vitro.

The term “compound” refers to any chemical entity, pharmaceutical, drug,and the like that can be used to treat or prevent a disease, illness,sickness, or disorder of bodily function. Compounds comprise both knownand potential therapeutic compounds. A compound can be determined to betherapeutic by screening using the screening methods of the presentinvention. A “known therapeutic compound” refers to a therapeuticcompound that has been shown (e.g., through animal trials or priorexperience with administration to humans) to be effective in suchtreatment. In other words, a known therapeutic compound is not limitedto a compound efficacious in the treatment of cancer.

The term “test compound” refers to any chemical entity, pharmaceutical,drug, and the like that can be used to treat or prevent a disease,illness, sickness, or disorder of bodily function. Test compoundscomprise both known and potential therapeutic compounds. A test compoundcan be determined to be therapeutic by screening using the methods ofthe present invention. A “known therapeutic compound” refers to atherapeutic compound that has been shown (e.g., through animal trials orprior experience with administration to humans) to be effective in suchtreatment or prevention. In other words, a known therapeutic compound isnot limited to a compound efficacious in the treatment of α7 instabilityor inactivity in animals.

A “composition comprising a given polynucleotide sequence” as usedherein refers broadly to any composition containing the givenpolynucleotide sequence. The composition may comprise an aqueoussolution. Compositions comprising polynucleotide sequences encodinghuman α7 (e.g., SEQ ID NO:123), or fragments thereof, may be employed ashybridization probes. In other embodiments, compositions comprising thepromoter and upstream untranslated sequence of human α7 (e.g., SEQ IDNO:122) or fragments thereof (e.g., SEQ ID NO:94, 101, 125, etc) may beemployed as hybridization probes. In these cases, the human α7-encodingpolynucleotide sequences are typically employed in an aqueous solutioncontaining salts (e.g., NaCl), detergents (e.g., SDS) and othercomponents (e.g., Denhardt's solution, dry milk, salmon sperm DNA,etc.).

The term “sample” as used herein is used in its broadest sense. A samplesuspected of containing a human chromosome or sequences associated witha human chromosome may comprise a cell, chromosomes isolated from a cell(e.g., a spread of metaphase chromosomes), genomic DNA (in solution orbound to a solid support such as for Southern blot analysis), RNA (insolution or bound to a solid support such as for Northern blotanalysis), cDNA (in solution or bound to a solid support) and the like.A sample suspected of containing a protein may comprise a cell, aportion of a tissue, an extract containing one or more proteins and thelike.

The term “test α7” refers to a sample suspected of containing α7. Theconcentration of α7 in the test sample is determined by various means,and may be compared with a “quantitated amount of α7” (i.e., a positivecontrol sample containing a known amount of α7), in order to determinewhether the concentration of test α7 in the sample is within the rangeusually found within samples from wild-type organisms. Thus, comparisonof the positive control with the test sample allows the determination tobe made whether a particular individual produces a “normal” amount ofα7, is deficient in production of α7, or produces a concentration of α7that is greater than normal. It is intended that such test methods alsocontain “negative” controls (i.e., samples that are known to contain noα7). Furthermore, it is intended that the testing be conducted using theα7 gene, α7 mRNA, and/or α7 protein (or polypeptides), or fragments ofany of these.

The term “heteroduplex analysis” as used herein refers to a method ofdetecting mutations based on the retardation of the heteroduplexcompared with the corresponding homoduplex on a non-denaturingpolyacrylamide gel. Heteroduplexes migrate more slowly than theircorresponding homoduplexes due to a more open double-strandedconfiguration surrounding the mismatched bases.

As used herein, the terms “DHPLC” and “denaturing high performanceliquid chromatography” refer to a scanning method for mutation detectionbased on the capability of ion-pair reverse phase liquid chromatographyon alkylated nonporous particles to resolve homo from heteroduplexmolecules under conditions of partial denaturation (Underhill et al.,Proc Natl Acad Sci USA, 93:196-2000, 1996 and U.S. Pat. No. 5,795,976,herein incorporated by reference in their entirety).

The terms “single-strand conformation polymorphism” and “SSCP,” as usedherein, refer to the ability of single strands of nucleic acid to takeon characteristic conformations under non-denaturing conditions, whichin turn can influence the electrophoretic mobility of thesingle-stranded nucleic acids. Changes in the sequence of a givenfragment (i.e., mutations) will also change the conformation,consequently altering the mobility and allowing this to be used as anassay for sequence variations (Orita et al., Genomics 5:874-879, 1989).

As used herein, the terms “conformation-sensitive gel electrophoresis”or “CSGE” refer to methods for detecting mutations involvingdistinguishing DNA heteroduplexes from homoduplexes via mildlydenaturing gel electrophoresis. CSGE protocols are well known in the art(Ganguly et al., Proc Natl Acad Sci USA 90:10325-10329, 1993).

As used herein, the terms “ligase chain reaction” and “ligaseamplification reaction” refer to methods for detecting small quantitiesof a target DNA, with utility similar to PCR. Ligase chain reactionrelies on DNA ligase to join adjacent synthetic oligonucleotides afterthey have bound the target DNA. Their small size means that they aredestabilized by single base mismatches and so form a sensitive test forthe presence of mutations in the target sequence.

The term “DNA sequencing” refers to methods used to determine the orderof nucleotide bases in a DNA molecule or fragment. The term “DNAsequencing” includes for example, dideoxy sequencing and Maxam-Gilbertsequencing.

As used herein, the term “in vitro” refers to an artificial environmentand to processes or reactions that occur within an artificialenvironment. In vitro environments can consist of, but are not limitedto, test tubes and cell culture. The term “in vivo” refers to thenatural environment (e.g., an animal or a cell) and to processes orreaction that occur within a natural environment.

The terms “test compound” and “candidate compound” refer to any chemicalentity, pharmaceutical, drug, and the like that is a candidate for useto treat or prevent a disease, illness (e.g., major depressivedisorder), sickness, or disorder of bodily function. Test compoundscomprise both known and potential therapeutic compounds. A test compoundcan be determined to be therapeutic by screening using the screeningmethods of the present invention.

The term “change” as used herein refers to a difference or a result of amodification or alteration. In preferred embodiments, the term “change”refers to a measurable difference between states (e.g., higher or lowerα7 mRNA or protein expression in a cell in the presence and absence of atest compound). In some embodiments, the change is at least 10%,preferably at least 25%, more preferably at least 50%, and mostpreferably at least 90% more or less than that of a control condition.

As used herein, the term “sample” is meant to include a specimenobtained from subject. The term “sample” encompasses fluids, solids, andtissues. In preferred embodiments, the term “sample” refers to blood orbiopsy material obtained from a living body for the purpose ofexamination via any appropriate technique (e.g., needle, sponge,scalpel, swab, etc.). In particularly preferred embodiments, the term“sample” refers to buccal cells (e.g., cells of the inner lining of themouth or cheek). Buccal cell samples are obtained using any suitablemethod, including but not limited to collection via tongue depressor,cytobrush or mouthwash (See, Moore et al., Biomarkers, 6:448-454, 2001).

The terms “subject” as used herein, refers to a human. It is intendedthat the term encompass healthy individuals, as well as, individualspredisposed to, or suspected of having schizophrenia. Typically, theterms “subject” and “patient” are used interchangeably. In somepreferred embodiments of the present invention, the term subject refersto specific subgroups of patients.

The term “schizophrenia” as used herein refers to a major mentaldisorder featuring psychotic symptoms during some phase of the illness,a long term course and a deterioration in function. Schizophrenicsymptoms can be classified as positive, negative, cognitive and moodsymptoms, which together or separately may result in behavioraldisturbances (e.g., bizarre, apparently purposeless and stereotypedactivity or inactivity). Various embodiments of the present inventionare contemplated to effectively treat all subtypes of schizophrenia,including but not limited to catatonic, disorganized, paranoid andundifferentiated subtypes. In addition, the compositions and methods ofthe present invention are also contemplated to benefit patients withschizoid personality disorder (socially distant, detached) and patientswith schizotypal personality disorder (odd, eccentric).

As used herein, the term “positive symptoms” refers to symptomsincluding but not limited to hallucinations (e.g., hearing voices),delusions (e.g., of persecution or grandiosity), disorganized speech andthought, altered sense of self and bizarre behavior. They are calledpositive symptoms because they are added on to the individualsexperience and behavior.

The term “negative symptoms” as used herein, refers to deficit symptoms,including experience and behavior that should be there and is not.Negative symptoms include but are not limited to loss of motivation,flattened emotions, withdrawal from an active social life, poverty ofthought and speech, and loss of former interests and pleasures.

As used herein, the term “cognitive symptoms,” refers to symptomsassociated with a loss of cognitive ability including but not limited toattention deficits, memory loss, inability to plan for the future andpoor capacity for abstract thought.

The term “mood symptoms” as used herein, refers to symptoms associatedwith a disturbed state of mind or predominant emotion such as dysphoria.

As used herein, the term “risk of developing schizophrenia” refers to asubject's relative risk (e.g., the percent chance or a relative score)of developing schizophrenia during their lifetime.

The term “subject suspected of having schizophrenia” refers to a subjectthat presents one or more symptoms indicative of schizophrenia (e.g.,delusions, hallucinations, disorganized speech, catatonic behavior,negative symptoms such as effective flattening, alogia or avolition,etc.) or is being screened for schizophrenia (e.g., during a routinephysical).

As used herein, the term “diagnosis” refers to the determination of thenature of a case of disease. In some preferred embodiments of thepresent invention, methods for making a diagnosis are provided whichpermit schizophrenia to be distinguished from other forms of mentalillness including but not limited to psychosis due to a general medicalcondition; delirium, or dementia; substance-induced or relateddisorders; depressive disorder; and bipolar disorder (e.g., manicdepression).

The term “reagent(s) suitable for use in specifically detecting at leastone polymorphism in an α7 allele” refers to reagent(s) used to detect apolymorphism of interest in an α7 gene, cDNA, or RNA. Examples ofsuitable reagents include but are not limited to, nucleic acid probesand primers capable of specifically hybridizing to α7 mRNA or cDNA. Insome preferred embodiments, the term suitable reagents refers to primersfor amplifying an α7 fragment suspected of containing a polymorphism ofinterest.

As used herein, the term “instructions for determining whether a subjectis predisposed to schizophrenia” refers to instructions for using thereagents contained in the kit for the detection and characterization ofan α7 allele in a sample from a subject. In some embodiments, theinstructions further comprise the statement of intended use required bythe U.S. Food and Drug Administration (FDA) in labeling in vitrodiagnostic products. The FDA classifies in vitro diagnostics as medicaldevices and required that they be approved through the 510(k) procedure.Information required in an application under 510(k) includes: 1) The invitro diagnostic product name, including the trade or proprietary name,the common or usual name, and the classification name of the device; 2)The intended use of the product; 3) The establishment registrationnumber, if applicable, of the owner or operator submitting the 510(k)submission; the class in which the in vitro diagnostic product wasplaced under section 513 of the FD&C Act, if known, its appropriatepanel, or, if the owner or operator determines that the device has notbeen classified under such section, a statement of that determinationand the basis for the determination that the in vitro diagnostic productis not so classified; 4) Proposed labels, labeling and advertisementssufficient to describe the in vitro diagnostic product, its intendeduse, and directions for use, including photographs or engineeringdrawings, where applicable; 5) A statement indicating that the device issimilar to and/or different from other in vitro diagnostic products ofcomparable type in commercial distribution in the U.S., accompanied bydata to support the statement; 6) A 510(k) summary of the safety andeffectiveness data upon which the substantial equivalence determinationis based; or a statement that the 510(k) safety and effectivenessinformation supporting the FDA finding of substantial equivalence willbe made available to any person within 30 days of a written request; 7)A statement that the submitter believes, to the best of their knowledge,that all data and information submitted in the premarket notificationare truthful and accurate and that no material fact has been omitted;and 8) Any additional information regarding the in vitro diagnosticproduct requested that is necessary for the FDA to make a substantialequivalency determination. Additional information is available at theInternet web page of the U.S. FDA.

DESCRIPTION OF THE FIGURES

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawings will be provided by the Patentand Trademark Office upon request and payment of the necessary fee.

FIG. 1 shows the exon-intron boundary sequences of the human α7 nAChRsubunit gene. The 3′ portion of exon 1 is disclosed as SEQ ID NO:84.Also shown are the splice acceptor sequences of: intron 1 (SEQ IDNO:85), intron 2 (SEQ ID NO:86), intron 3 (SEQ ID NO:87), intron 4 (SEQID NO:88), intron 5 (SEQ ID NO:89), intron 6 (SEQ ID NO:90), intron 7(SEQ ID NO:91), intron 8 (SEQ ID NO:92) and intron 9 (SEQ ID NO:93), aswell as the splice donor sequences of: intron 1 (SEQ ID NO:104), intron2 (SEQ ID NO:106), intron 3 (SEQ ID NO:108), intron 4 (SEQ ID NO:110),intron 5 (SEQ ID NO:112), intron 6 (SEQ ID NO:114), intron 7 (SEQ IDNO:116), intron 8 (SEQ ID NO:118), and intron 9 (SEQ ID NO:120).Additionally, flanking exon sequences are shown: exon 2 (SEQ ID NO:105),exon 3 (SEQ ID NO:107), exon 4 (SEQ ID NO:109), exon 5 (SEQ ID NO:111),exon 6 (SEQ ID NO:113), exon 7 (SEQ ID NO:115), exon 8 (SEQ ID NO:117),exon 9 (SEQ ID NO:119), and exon 10 (SEQ ID NO:121).

FIG. 2 shows the sequence variants identified in full-length andduplicated genomic clones.

FIG. 3 provides an expression analysis of sequence variants.

FIG. 4 provides the nucleotide sequence of the region 5′ of the human α7nAChR subunit gene (SEQ ID NO:94).

FIG. 5 shows the genomic contig of clones positive for α7 nAChR genesequences and surrounding loci.

FIG. 6 provides a partial sequence of a RACE clone, with exon sequencesshown in upper case and intron sequences shown in lower case: exon D(SEQ ID NO:95), exon C (SEQ ID NO:96), exon B (SEQ ID NO:97), exon A(SEQ ID NO:98), exon 5 (SEQ ID NO:99), and exon 6 (SEQ ID NO:100).

FIG. 7 provides a map of the full-length α7 nAChrR gene. Panel A shows aphysical map of the region and the structure of the full-length α7 nAChRgene. Panel B shows the structure of alternatively spliced transcripts.

FIG. 8 provides the DNA sequence of the human α7 neuronal nicotinicreceptor promoter (SEQ ID NO:101).

FIG. 9 provides the DNA sequence of the alternatively spliced human α7neuronal nicotinic receptor RACE product A/C/D (SEQ ID NO:102).

FIG. 10 provides the DNA sequence of the alternatively spliced human α7neuronal nicotinic receptor RACE product A/B/C/D (SEQ ID NO:103).

FIG. 11 provides a physical map of the linkage region to schizophreniaon chromosome 15q13-q14. The estimated size of the region is 4 cm.

FIG. 12 depicts the promoter region of the α7 nicotinic acetylcholinereceptor gene (CHRNA7). Panel A shows the fragments used to identify thecore promoter region. Panel B shows the core promoter region for theCHRNA7 gene set forth as SEQ ID NO:125. Arrows depict the locations ofpolymorphisms identified with the mutation screen.

FIG. 13 provides the results of a functional assay of the α7 corepromoter variants. The activity of the normal promoter sequence was setat 100%. Symbols are indicative of the following P values: asterisk,P<0.0001; dagger, P=0.005; double dagger, P=0.05; and section mark,P=0.03.

FIG. 14 shows the gating of the P50 auditory evoked potential in controlsubjects (Panels A and B), and in a schizophrenic subject (Panel C).Tracings are shown for both the conditioning and test responses. Arrowsindicate the paired auditory stimuli.

FIG. 15 depicts the association between promoter variants and P50 gatingin control subjects. In Panel A, mean P50 ratios are shown for controlsubjections without (N/N) and with one or more polymorphisms (Poly) inthe α7 core promoter. In Panel B, promoter variants in control subjectsare shown to fit into three P50 gating ranges.

FIG. 16 provides a schematic of the 15q13-q14 region containing theCHRNA7 and dupCHRNA7 genes in Panel A. The transcripts from both the α7containing genes are shown with their unique 5′ ends in Panel B. Thenumber of variants mapped to each exon is shown in parentheses.

FIG. 17 depicts predictive patterns for 24 different mutations in theCHRNA7 proximal promoter determined through use of a Transgenomics WAVE™denaturing high performance liquid chromatography (DHPLC) system. Thepatterns are as follows: (A) wild type; (B) −194 G/C; (C) −86 C/T; (D)−46 T; (E) −46 G/T; (F) −92 G/A; (G) −143 G/A; (H) −166 C/T; (I) −178−G;(J) −180 G/C; (K) −190+G; (L) −191 G/A; (M) −140+CGGG; (N) −178+CGGGGG;(O) −241 A/G; (P) −46 G/T and −178−G; (Q) −46 G/T and −190+G; (R) −46G/T and −191 G/A; (S) −46 G/T and −194 G/C; (T) −86 C/T and −194 G/C;(U) −86 C/T and −241 A/G; (V) −93 C/G and −194 C/G; (W) −178−G and−190+G; (X)−178−G and −191 G/A; and (Y) −191 G/A and −194 G/C.

FIG. 18 presents several embodiments of polymorphisms identified in theCHRNA7 proximal promoter and 5′-upstream regulatory region (SEQ ID NO:181). Polymorphisms identified are in red. The polymorphism associatedwith schizophrenia in the current study is highlighted in yellow.Transcription factor binding sites are indicated by blue bars.

FIG. 19 presents exemplary data using a Haploview graph of the pairwiselinkage disequilibrium (LD) between SNPs in the proximal promoter ofCHRNA7, and SNPs in the upstream regulatory region of the gene. A)African-American sample population and B) Caucasian sample population.Numbers inside the squares are D′×100.

FIG. 20 presents illustrative amplicon embodiments useful for thedetection and identification of polymorphisms.

DESCRIPTION OF THE INVENTION

The present invention is related to the alpha-7 neuronal nicotinicacetylcholine receptor gene. In particular, the present inventionprovides the human alpha-7 gene. In addition, the present inventionprovides methods and compositions for the diagnosis and treatment ofschizophrenia. Such compositions include, but are not limited to,polymorphisms within the human alpha-7 gene promoter core and/or 5′upstream regulatory regions.

As the methods of the prior art have been unsuccessful in providingneeded information regarding the genetics of schizophrenia, analternative strategy for genetic studies of complex diseases involvingthe use of a specific neurobiological characteristic of the illness(e.g., as an additional phenotype more closely reflecting the effect ofa single genetic alteration), was used during the development of thepresent invention. Such information is needed in order to providediagnostic and treatment methods, as well as animal models forschizophrenia, as well as other psychoses. It is contemplated that sucha trait is part of the inherited diathesis of the illness, whichproduces schizophrenia in combination with other pathogenic elements.

The present invention provides genetic information (i.e., sequences,including sequence location and information on intron/exon boundaries)for the α7 nicotinic receptor, as well as methods to assess the functionof this receptor in normal, as well as schizophrenic individuals. Thepresent invention also provides methods and compositions for analyzingsamples from patients suspected of suffering from diverse conditions,including epilepsy (e.g., juvenile myoclonic epilepsy), small cell lungcarcinoma and other nicotine-dependent diseases, Prader-Willi,Angelman's syndrome, and other genetic disorders, etc. Indeed, it is notintended that the present invention be limited to schizophrenia.

I. Schizophrenia and Smoking

Schizophrenia is a complex neuropsychiatric disorder partiallycharacterized by sensory processing and cognitive deficits. Leonard etal., “Smoking and mental illness” Pharmacol. Biochem. Behav. 70:561-570(2001); Light et al., “Sensory gating deficits in schizophrenia: can weparse the effects of medication, nicotine use, and changes in clinicalstatus?” Clin. Neurosci. Res. 3:47-54 (2003); George et al., “Apreliminary study of the effects of cigarette smoking on prepulseinhibition in schizophrenia: involvement of nicotinic receptormechanisms” Schizophr. Res. 87:307-315 (2006); Adams et al., “Evidencefor a role of nicotinic acetylcholine receptors in schizophrenia” Front.Biosci. 12:4755-4772 (2007); and Martin et al., “Schizophrenia and thealpha7 nicotinic acetylcholine receptor” Int. Rev. Neurobiol. 78:225-246(2007). Sensory processing deficits may be normalized in schizophrenicpatients and first-degree relatives by smoking. Adler et al.,“Normalization of auditory physiology by cigarette smoking inschizophrenic patients” Am. J. Psych. 150:1856-1861 (1993); Adler etal., “Schizophrenia, sensory gating, and nicotinic receptors” Schizofr.Bull. 24:189-202 (1998); Olincy et al., “Improvement in smooth pursuiteye movements after cigarette smoking in schizophrenic patients”Neuropsychopharmacology 18:175-185 (1998); and Leonard et al., “Geneticsof smoking and schizophrenia” J. Dual Diag. 3:43-59 (2007). Further, ithas been reported that cognitive deficits may be improved by nicotineadministration. Levin et al., “Transdermal nicotine effects onattention” Psychopharmacology 140:135-141 (1998); Levin et al.,“Nicotinic effects on cognitive function: behavioral characterization,pharmacological specification, and anatomic localization”Psychopharmacology 184:523-539 (2006); and Rezvani et al., “Cognitiveeffects of nicotine” Biol. Psychiatry 49:258-267 (2001).

Consequently, it has been suggested that schizophrenics may smoke to‘self-medicate’, thereby correcting an underlying biological deficit.Leonard et al., “Consequences of low levels of nicotinic acetylcholinereceptors in schizophrenia for drug development” Drug Dev. Res.60:127-136 (2003); Leonard et al., “Smoking and schizophrenia: evidencefor self medication” Schizophr. Bull. 33:262-263 (2007); and Kumari etal., “Nicotine use in schizophrenia: the self-medication hypotheses”Neurosci. Biobehav. Rev. 29:1021 (2005). This hypothesis has beeninvestigated in studies demonstrating that smoking alters geneexpression in normal individuals and differentially regulates geneexpression in schizophrenics. Mexal et al., “Differential modulation ofgene expression in the NMDA postsynaptic density of schizophrenic andcontrol smokers” Mol Brain Res. 139:317-332 (2005); Mexal et al.,“Regulation of a novel alphaN-catenin splice variant in schizophrenicsmokers” Am. J. Med. Genet. B Neuropsychiatr. Genet. 147B:759-768(2008); 2008; and Kuehn, “Link between smoking and mental illness maylead to treatments” J. Am. Med. Assoc. 295:483-484 (2006).

Nicotine is believed to exert its effect through neuronal nicotinicacetylcholine receptors expressed in the brain and periphery. Leonard etal., “Neuronal nicotinic receptors: from structure to function” NicotineTob. Res. 3:203-223 (2001); and Gotti et al., “Brain nicotinicacetylcholine receptors: native subtypes and their relevance” TrendsPharmacol. Sci. 27:482-491 (2006). The α7 neuronal nicotinic receptorsubunit gene (CHRNA7), localized at 15q14, was first genetically linkedto the P50 auditory sensory processing deficit in schizophrenia (infra).Freedman et al., “Linkage of a neurophysiological deficit inschizophrenia to a chromosome 15 locus” Proc. Natl. Acad. Sci. USA94:587-592 (1997). Subsequently, the gene was linked to the developmentand expression of schizophrenia. For example, patient cohorts ofEuropean American, African American, South African Bantu, Azorean, andHan Chinese ancestry, indicated that the linkage may be observed acrossethnicities. Coon et al., “Genomic scan for genes predisposing toschizophrenia” Am. J. Med. Genet. 54:59-71 (1994); Kaufmann et al.,“NIMH Genetics Initiative Millenium Schizophrenia Consortium: linkageanalysis of African-American pedigrees” Am. J. Med. Genet. 81:282-289(1998); Leonard et al., “Further investigation of a chromosome 15 locusin schizophrenia: analysis of affected sibpairs from the NIMH GeneticsInitiative” Am. J. Med. Genet. 81:308-312 (1998); Riley et al.,“Haplotype transmission disequilibrium and evidence for linkage of theCHRNA7 gene region to schizophrenia in Southern African Bantu families”Am. J. Med. Genet. 96: 196-201 (2000); Freedman et al., “Linkagedisequilibrium for schizophrenia at the chromosome 15 q13-14 locus ofthe alpha 7-nicotinic acetylcholine receptor subunit gene (CHRNA7)” Am.J. Med. Genet. 105:20-22 (2001); Gejman et al., “Linkage analysis ofschizophrenia to chromosome 15” Am. J. Med. Genet. 105:789-793 (2001);Liu et al., “Suggestive evidence for linkage of schizophrenia to markersat chromosome 15q13-14 in Taiwanese families” Am. J. Med. Genet.105:658-661 (2001); Tsuang et al., “Examination of genetic linkage ofchromosome 15 to schizophrenia in a large veterans affairs cooperativestudy sample” Am. J. Med. Genet. 105:662-668 (2001); Xu et al.,“Evidence for linkage disequilibrium between the alpha 7-nicotinicreceptor gene (CHRNA7) locus and schizophrenia in Azorean families” Am.J. Med. Genet. 105:669-674 (2001); and Fallin et al., “Genome-widelinkage scan for schizophrenia susceptibility loci among AshkenaziJewish families shows evidence of linkage on chromosome 10q22” Am. J.Hum. Genet. 73:601-611 (2003).

Activation of the homopentameric α7* receptor has been reported toresult in the influx of Ca²⁺ and concomitant neurotransmitter release.Vijayaraghavan et al., “Nicotinic receptors that bind α-bungarotoxin onneurons raise intracellular free Ca⁺⁺” Neuron 8: 353-362 (1992);Aramakis et al., “Nicotine selectively enhances NMDA receptor-mediatedsynaptic transmission during postnatal development in sensory neocortex”J. Neurosci. 18: 8485-8495 (1998); Berg et al., “Nicotinic alpha 7receptors: synaptic options and downstream signaling in neurons” J.Neurobiol. 53:512-523 (2002); Dajas-Bailador et al., “Nicotinicacetylcholine receptors and the regulation of neuronal signalling”Trends Pharmacol. Sci. 25:317-324 (2004). Binding studies with an α7*receptor antagonist (i.e., for example, α-bungarotoxin) show there are50% fewer receptors in post-mortem hippocampus of individuals withschizophrenia compared to control subjects. Freedman et al., “Evidencein postmortem brain tissue for decreased numbers of hippocampalnicotinic receptors in schizophrenia” Biol. Psychiatry 38:22-33 (1995).Decreased α7* receptor expression was also found in cortex and in thereticular thalamic nucleus. Guan et al., “Decreased protein level ofnicotinic receptor alpha7 subunit in the frontal cortex fromschizophrenic brain” Neuroreport 10:1779-1782 (1999); Marutle et al.,“Laminar distribution of nicotinic receptor subtypes in cortical regionsin schizophrenia” J. Chem. Neuroanat. 22:115-126 (2001); and Court etal., Neuronal nicotinic receptors in dementia with Lewy bodies andschizophrenia: alpha-bungarotoxin and nicotine binding in the thalamus”J. Neurochem. 73:1590-1597 (1999), respectively. Although it is notnecessary to understand the mechanism of an invention, it is believedthat low levels of the α7* receptor may have downstream consequences formultiple neurotransmitter systems, thereby altering the balance ofneurotransmitter release and activation. Leonard S., “Consequences oflow levels of nicotinic acetylcholine receptors in schizophrenia fordrug development” Drug Dev. Res. 60:728:127-136 (2003).

The CHRNA7 gene has been suggested as a candidate gene forschizophrenia. Harrison et al., “Schizophrenia genes, gene expression,and neuropathology: on the matter of their convergence” Mol. Psychiatry10:40-68 (2005). Further, α7* receptor agonists have been identified asputative targets for drug development to treat schizophrenic cognitiveimpairments. Psychiatric News, Molecular Targets Ranked. Psych. News41:16-1a (2006). Recent Phase I and Phase II studies of an α7* receptorpartial agonist, DMXB-A, resulted in improvements of both sensoryprocessing and attention in non-smoking schizophrenics. Martin et al.,“Alpha-7 nicotinic receptor agonists: potential new candidates for thetreatment of schizophrenia” Psychopharmacology 174:54-64 (2004); Olincyet al., “Proof-of-concept trial of an alpha 7 nicotinic agonist inschizophrenia” Arch. Gen. Psychiatry 63: 630-638 (2006); Freedman etal., “Initial phase 2 trial of a nicotinic agonist in schizophrenia” Am.J. Psychiatry (submitted, 2008).

The α7 nicotinic receptor subunit gene, CHRNA7, was found to bepartially duplicated, with both loci mapping to the 15q14 region. Gaultet al., “Genomic organization and partial duplication of the human α7neuronal nicotinic acetylcholine receptor gene” Genomics 52:173-185(1998); Riley et al., “A 3 Mb map of a large segmental duplicationoverlapping the alpha 7-nicotinic acetylcholine receptor gene (CHRNA7)at human 15q13-q14” Genomics 79:197-209 (2002). The partially duplicatedgene (CHRFAM7A) is believed located 1.6 Mb centromeric to thefull-length gene, however, its function remains unknown. Mutationscreening of the coding region and intron/exon borders of CHRNA7 and ofCHRFAM7A has identified thirty-three (33) putative polymorphisms. Three(3) of the polymorphisms were non-synonymous and mapped to the fulllength CHRNA7 coding region. These polymorphisms, however, were veryrare and were not associated with either schizophrenia or the P50112340123456789 gating deficit. Gault et al., “Comparison ofpolymorphisms in the α7 nicotinic receptor gene and its partialduplication in schizophrenic and control subjects” Am. J. Med. Genet.123B:39-49 (2003).

In contrast, mutation screening of the core promoter in the CHRNA7 geneidentified a large number of polymorphisms predictive of schizophrenia.For example, a functional analysis of polymorphisms in the two hundredand thirty-one (231) base pairs upstream of the α7* translationinitiation site (i.e., for example, the core promoter region)demonstrated that many of the identified polymorphisms decrease α7*receptor transcription. These polymorphisms were also found to bestatistically more prevalent in schizophrenics than in control subjects(P=0.007). Further, the presence of a promoter polymorphism innon-schizophrenic controls was associated with a P50 gating deficit(P<0.0001). Reporter gene assays with fragments 1.0 kb and 2.6 kbproximal to the translation initiation site exhibit less activity thanthe core promoter, suggesting that repressor elements may lie upstreamof the core promoter. Leonard et al., “Association of promoter variantsin the alpha 7 nicotinic acetylcholine receptor subunit gene with aninhibitory deficit found in schizophrenia” Arch. Gen. Psychiatry59:1085-1096 (2002).

I. Inhibition of the P50 Auditory Response

Various psychophysiological paradigms demonstrate altered brainfunctions in schizophrenic patients and their relatives that mightreflect inherited traits (See e.g., Sham et al., Psychiat Genet, 4:29,1994; De Amicis et al., J Nerv Ment Dis, 174:177, 1986; Holzman et al.,Arch Gen Psychiat, 45:641, 1988; and Braff et al., Arch Gen Psychiat,49:206, 1992). Basic deficits in the regulation of response to sensorystimuli may underlie patients' more apparent symptoms such ashallucinations and delusions. In addition to hearing voices, patientsoften attend to apparently extraneous stimuli in their surroundings thatnormal individuals generally ignore. Such symptoms suggested thatneuronal mechanisms responsible for the filtering or gating of sensoryinput to higher brain centers are deficient. One method developed forexamining such neuronal mechanisms compares the responses to first andsecond of paired stimuli. The first stimulus elicits an excitatoryresponse that also activates inhibitory mechanisms, which then diminishthe excitatory response to the second stimulus. The ratio of theamplitude of the second response to the first is inversely related tothe strength of inhibition.

During the development of the present invention, this method was used tostudy the response to auditory stimuli in schizophrenia, using anelectrically positive evoked potential occurring 50 ms after an auditorystimulus (P50). Inhibition of the P50 response to a second identicalstimulus (presented 500 ms after the first) has been reported to bediminished in schizophrenics (Adler et al., Biol Psychiat, 17:639, 1982;Boutros and Overall, Clin Electroencephalog, 22:20, 1991; Erwin et al.,Biol Psychiat, 30, 430, 1991; and Judd et al., Am J Psychiat, 149:488,1992). This diminished inhibition, measured as an elevation in the ratioof P50 amplitudes, has been correlated with schizophrenics' decreasedperformance in a neuropsychological measure of sustained attention, aswell as diminished performance in a word recognition task (Cullum etal., Schizophrenia Res, 10:131, 1993; and Vinogradova et al., BiolPsychiat, 39: 821, 1996).

In the development of the present invention, inhibition of the P50response was measured in animal and related clinical investigations, toidentify neurobiological mechanisms related to genes of interest, aswell as a phenotype for linkage analysis to identify chromosomal areascontaining genes responsible for the abnormality in schizophrenics.

The neurobiological inhibition of human P50 to repeated auditory stimuliwas initially investigated using an auditory evoked potential recordedfrom the rat as a model. Both the human and rat potentials show similardecreased responses to repeated auditory stimuli (Adler et al., BiolPsychiat, 21:787, 1986). Neuronal recordings identified the pyramidalneurons of the hippocampus as a major source of the rat evokedpotential. These pyramidal neurons have a decremented response torepeated auditory stimuli that parallels the decrement in the evokedpotential (Bickford-Wimer et al., Biol Psychiat, 27:183, 1990). Thedecrement is lost after transfection of the fimbria-fornix, a fibertract that includes afferents to the hippocampus from cholinergicneurons in the basal forebrain (Vinogradova, in The Hippocampus 2:Neurophysiology and Behavior, Issacson and Pribram (eds), Plenum Press,New York, N.Y., pp 3-69, 1975).

However, nicotine has been found to normalize inhibition of response inthe fimbria-fornix lesioned animals (See e.g., Bickford and Wear, BrainRes, 705:235, 1995). Studies with pharmacological antagonists inunlesioned animals indicate that a specific subset of nicotiniccholinergic receptors is involved in the inhibitory mechanism. Theinhibition is selectively blocked by the snake toxin α-bungarotoxin(Luntz-Leybman et al., Brain Res, 587:130, 1992), suggesting that thereceptor contains the α7 nicotinic cholinergic receptor subunit, as itis the only known nicotinic receptor subunit in the mammalian brainsensitive to this toxin (Couturier et al., Neuron, 5:847-856, 1990; andSchoepfer et al., Neuron, 5:35, 1990). Neither scopolamine, normecamylamine, nor α-bungarotoxin (i.e., antagonists of other types ofcholinergic receptors), blocked the inhibition. Receptor autoradiographyusing [125I]-α-bungarotoxin showed the most intense binding tonon-pyramidal hippocampal neurons containing the inhibitoryneurotransmitter γ-aminobutyric acid (Freedman et al., J Neurosci,13:1965, 1993). This labeling was consistent with physiological evidencethat cholinergic synapses activate interneurons, which inhibit thepyramidal neuron response to the second stimulus (See e.g., Miller andFreedman, Neurosci, 69:371-381, 1995; and Hershman et al., NeurosciLett, 190:133, 1995).

There are several areas of apparent concordance between these findingsin rats and P50 inhibition in humans. First, P50 has been recorded fromthe human hippocampus (Goff et al., Prog Clin Neurophysiol, 7:126, 1980;and Makela et al., Electroencephalogr Clin Neurophysiol, 92:414, 1994),and human hippocampal neurons have rapidly decreasing responses toauditory stimuli, similar to those observed with rat hippocampal neurons(Wilson et al., Exp Neurol, 84:74, 1984). Second, nicotine in high dosestransiently normalizes the abnormality in P50 inhibition inschizophrenics and in their relatives, much as it normalizes inhibitionin rats after fimbria-fornix lesions (Bickford and Wear, supra, 1995;Adler et al., Biol Psychiatry, 32:607, 1992; and Adler et al., Am JPsychiat, 150:1856, 1993). However, the effect of nicotine on P50inhibition in relatives of schizophrenics is not blocked bymecamylamine, which blocks all known nicotinic receptors in human brain,except the α7 nicotinic receptor (Freedman et al, Harvard Rev Psychiat,2:179, 1994). In situ hybridization has shown that α7 nicotinic receptormRNA is expressed in human hippocampal neurons (Freedman et al., HarvardRev Psychiat, 2:179, 1994). Some of the non-pyramidal neurons of thehuman hippocampus were intensely labeled by α-bungarotoxin, as was alsoobserved with rats.

A preliminary study showed that α-bungarotoxin labeling was decreased inpost mortem hippocampus from eight schizophrenics (Freedman et al., BiolPsychiat, 38:22, 1995). In addition, schizophrenic patients areparticularly heavy tobacco smokers, even when compared to otherpsychiatric patients (deLeon et al., Am J Psychiat, 152:453, 1995; andHamera et al., J Nerv Mental Dis, 183:559, 1995). This heavy nicotineuse may reflect an attempt at self-medication of an endogenous neuronaldeficit (Goff et al., Am J Psychiat, 149:1189, 1992). However,nicotine's efficacy as an anti-psychotic is limited, due to rapiddesensitization and cardiovascular toxicity.

In parallel with these biological studies in human and animals, the P50evoked potential abnormality was also investigated as a phenotype forgenetic linkage analysis. A genome-wide scan was initiated, independentof any candidate gene hypothesis, in nine multiplex schizophrenicpedigrees, which were also phenotyped with P50 recordings. The deficitin inhibition of the P50 response in these and other schizophrenicfamilies is generally found in one of the parents and half the siblings,including the schizophrenic probands (Siegel et al., Arch Gen Psychiat,41:607-612, 1984). Although elevated P50 ratios are significantlyassociated with the apparent genetic risk for schizophrenia, manyindividuals in the pedigrees who have the deficit are clinicallyunaffected (Waldo et al., Psychiat Res, 39:257, 1991). In this respect,the distribution of the trait resembles several other neurobiologicalabnormalities in schizophrenics and their relatives, such as deficits insmooth pursuit eye movements and reaction time (De Amicis et al., J NervMent Dis, 174:177, 1986; and Holzman et al. Arch Gen Psychiat, 45:641,1988). These traits may represent alternative expressions of a latenttrait or endophenotype, which, in combination with other pathogenicelements, gives rise to schizophrenia.

During the development of the present invention, preliminary linkageanalyses between the P50 ratio abnormality and 318 restriction fragmentlength polymorphism and tandem repeat DNA markers in the nine kindredswere conducted. DNA markers mapping to four chromosomal regions, one ofwhich was 15q14, revealed small positive lod scores (maximum logarithmof the odds) assuming autosomal dominant transmission. Subsequently, theα7 nicotinic receptor gene was localized to the 15q14 region(Orr-Urtreger et al., Genomics, 26:399, 1995; and Spitzer et al., ArchGen Psychiat, 35:773, 1978). As converging evidence from neurobiologicalinvestigations implicated α7 receptor function in abnormal P50inhibition, and the preliminary linkage study provided suggestiveevidence for heritability of the trait near the chromosomal location ofthe α7 receptor gene, additional experiments, using informative markersat the α7 receptor gene locus were undertaken. Two new DNA polymorphicloci were isolated, namely D15S1360 from a yeast artificial chromosome(YAC) containing the α7 nicotinic receptor gene, and L76630 from anα7-containing clone in a genomic phage library. These markers were usedwith over 500 highly polymorphic markers in a 10 centiMorgan resolutiongenome-wide scan of the nine pedigrees. The results demonstrate a highlysignificant linkage between D15S1360 and the abnormality in P50suppression.

Indeed, the data obtained during the development of the presentinvention strongly suggest that the P50 auditory sensory deficit inschizophrenia is genetically linked to the locus of the α7 nicotinicreceptor gene on chromosome 15q14. Thus, the present invention providesa heretofore unknown linkage between nicotinic receptors andschizophrenia. The significant linkage obtained with the P50 ratiophenotype supports the value of this strategy. This provides methods forassessing the effects of therapy to correct abnormalities in α7structure and/or function, as well as providing methods for developingand identifying drugs suitable for use in treating such abnormalities.

Although an understanding of the mechanism is not necessary in order touse the present invention, it has been suggested that the clinicalillness may be less penetrant, because multiple genetic and non-geneticfactors are required to produce clinical illness, whereas a specificbiological defect may occur as the result of a single gene effect. Thus,some gene carriers would be expected to have abnormal P50 ratio, themore penetrant phenotype, but not schizophrenia, which is lesspenetrant. The lower lod scores observed during the development of thepresent invention with schizophrenia as a phenotype support thatposition; several kindreds had higher lod scores for P50 ratio than forschizophrenia because there were many family members with abnormal P50ratios who did not have schizophrenia.

The possibility that the chromosome 15q13-14 region is involved inpsychotic illness has also been investigated in relationship to otherdiseases. For example, psychoses resembling schizophrenia have beenobserved in Prader-Willi syndrome, a mental retardation linked todeletions and abnormal DNA imprinting in the 15 q11-13 region (Clarke,Brit J Psychiat, 163:680, 1993). The imprinting abnormality affects theexpression of many genes in this region. Several families in Sephardicand other populations have co-existent schizophrenia and Marfan'ssyndrome (i.e., a disease linked to dominant mutations in the fibrillingene at 15q21; Sirota et al., Br J Psychiat, 157: 433, 1990; andMelissari et al., Pathologica, 87:78, 1995). The co-segregation of thetwo illnesses may be based on their chromosomal proximity. Psychosis wasalso observed in a large French-Canadian kindred that has a recessivedemyelination disease, linked to markers at 15q14 (Casaubon et al., Am JHum Genet, 58:28, 1996). In addition, an Italian kindred contains twocousins with psychotic illness and a partial trisomy of chromosome 15,derived independently from abnormal meioses involving a balancedfamilial translocation with a 15q13 breakpoint, that was present in eachof their mothers. It was suggested that the new trisomies may havecaused the de novo appearance of illness (Calzolari et al., Am J MedGenet, 67:154, 1996). The present invention provides the means todetermine to what extent the appearance of psychoses in these familieswith other genetic abnormalities at 15q13-14 involves the α7 gene.

In addition to providing means to assess the risk for development ofschizophrenia, the present invention also provides new data about theidentity of neuronal abnormalities involved in its pathophysiology, aswell as the means to develop treatment methods and compounds, diagnosticmethods and reagents, and models (e.g., cell lines and transgenicanimals) of these neuronal abnormalities. These results are consistentwith clinical and neurobiological evidence for the involvement of the α7nicotinic receptor gene in sensory gating deficits in schizophrenia.

The present invention also provides the means to determine the role theα7 receptor in the sensory processing defects and other abnormalities inschizophrenia. The finding of a significant linkage supporting the roleof the α7 nicotinic receptor in the pathophysiology of sensory andattentional disturbance in schizophrenia, is unique. Manyneurotransmitter systems have been hypothesized to be at least partlyresponsible for schizophrenia, but direct biological assessment of aspecific neuronal receptor function in human subjects is generally notfeasible because of the brain's complexity and inaccessibility. Thepresent invention provides compositions and methods to overcome thesedrawbacks. Genetic investigations, including linkage studies, haverepresented the critical test of the involvement of a particularmechanism in schizophrenia. The present invention provides methods andcompositions to complement and/or replace such tests for schizophrenia.Indeed, linkage at the α7 nicotinic receptor locus thus supports theneurobiological evidence that this gene plays a role in apathophysiological aspect of schizophrenia, a role that prior to thepresent invention, had not been previously considered nor described,despite schizophrenics' well-known heavy dependence on nicotine.

II. Neuronal Nicotinic Receptor Subunit Family

As discussed above, during the development of the present invention, theα7 nicotinic receptor was associated with pathophysiological aspect(s)of schizophrenia. This receptor is a member of the neuronal nicotinicreceptor subunit gene family, which is expressed in mammalian brain aspentameric, ligand-gated ion channels (Patrick et al., Ann NY Acad Sci,505:194, 1987; Cooper et al., Nature, 350:235, 1991; and Lindstrom etal., Ann NY Acad Sci, 757:100, 1996). In the muscle, five differenttypes of subunits constitute the holoreceptor, but in brain only twotypes of subunits, designated as “α” and “β,” have been found (Galzi etal., Ann Rev Pharmacol, 31:37, 1991; and Lukas and Bencherif, Int RevNeurobiol, 34:25, 1992).

Neuronal receptors can be functionally differentiated into two principalclasses, which differ in their affinity for nicotine and the snaketoxin, α-bungarotoxin (Marks and Collins, Mol Pharmacol, 22:554, 1982;Wonnacott, J Neurochem, 47:1706, 1986; Marks et al., Mol Pharmacol,30:427, 1986; and Amar et al., FEBS, 327:284, 1993). Receptors that bindnicotine with high affinity contain α2-α6 as ligand binding subunits andrequire an association with β subunits for functional expression(Goldman et al., Cell, 48:965, 1987; Deneris et al., Clin Chem, 35:731,1989; and Wada et al., J Compar Neurol, 284:314, 1989). A second classof receptors (α7-α9) bind nicotine with low affinity, have a highaffinity for α-bungarotoxin, and function as homomeric ion channels inin vitro expression systems (Marks et al., supra, 1986; Wonnacott,supra, 1986; Alkondon and Albuquerque, J Pharm Ex Ther, 265:1455, 1993;Amar et al., FEBS, 327:284, 1993; and Zhang et al., Neuron, 12:167,1994). The α7 receptor is the only α-bungarotoxin-binding receptoridentified in mammalian brain. The α8 receptor appears to be onlyexpressed in chick (Schoepfer et al., Neuron, 5:35, 1990), and the α9receptor has limited expression in cochlear hair cells and pituitary(Elgoyhen et al., Cell, 79:705, 1994). In addition, a cDNA clone of thehuman α7 was isolated from a human brain library (GenBank Accession No.U40583).

Expression and function of a specific subset of the nicotinic receptorfamily, the α7 receptor, has recently been implicated in a neuronalpathway controlling the filtering or gating of auditory stimuli in bothhuman and rat brain (Adler et al., Biol Psych, 32:607, 1992; Adler etal., Am J Psychol, 150:1856, 1993; Freedman et al., Harvard RevPsychiat, 2:179, 1994; and Leonard et al., supra 1996). This sensoryprocessing mechanism is aberrant in a majority of subjects withschizophrenia (Freedman et al., Schiz Res, 4:233-243, 1991).Pharmacological studies in both humans and rats suggest that the deficitin humans can be normalized by nicotine (Adler et al., supra, 1992; andAdler et al., supra 1993) and reproduced in a rodent model byantagonists of the low affinity α7 nicotinic receptor but not by highaffinity antagonists (Luntz-Leybman et al., Brain Res, 587:130, 1992;and Rollins et al., Soc Neurosci Abst, 22:1272, 1996). Expression ofα-bungarotoxin binding receptors is decreased in schizophrenichippocampi by approximately 40% (Freedman et al., Biol. Psychiat, 38:22,1995).

During the development of the present invention, the locus D15S1360, apolymorphic marker <120 kb from the full-length α7 nicotinic receptorgene at 15q14, was genetically linked to this auditory gating deficit inschizophrenic pedigrees. However, it is contemplated that other genesmapping to the 15q14 region are potential alternative or additionalgenetic candidates to α7 for pathogenic features of schizophrenia.

Also during the development of the present invention, expression of theα7 nicotinic receptor was examined in human postmortem brain. This genewas widely expressed in most nuclei, albeit at low levels. Regions ofhighest expression included those involved in processing of sensoryinformation, such as the hippocampus, lateral and medial geniculates,and the reticular nucleus of the thalamus.

The present invention further provides the physical mapping of afull-length human genomic clone for the α7 receptor subunit andsequencing of a putative promoter region. The gene and promoterstructure are similar to that of the chick α7. Additionally, a partialα7 gene duplication including exons 5-10 and intervening intronicsequence, which lies <1 Mb from the full-length gene has beenidentified. In addition, four novel exons at the 5′ end of theduplicated α7 sequences were sequenced and intron/exon junctionsidentified. The duplicated α7 sequences were found to be expressed asalternatively spliced transcripts containing some or all of these novelexons.

The present invention also provides the structural organization of thehuman α7 neuronal nicotinic acetylcholine receptor gene, and presentsdata indicating a partial gene duplication. Large insert genomic cloneswere isolated from YAC, BAC and PAC libraries. There are 10 exons in thegene; the splice junctions are consistent with consensus splice sites(Green, Ann Rev Cell Biol, 7:559, 1991; Lamond, Bioessays, 15:595, 1993)and have an identical location to those in the chick α7 gene(Matter-Sadzinski et al., EMBO J, 11:4529, 1992), the only species forwhich genomic α7 gene sequence has been previously published.

The promoter region of the gene was found to be 77% G/C, and contains noTATA box. It thus fits a growing group of eukaryotic promoters, whichdemonstrate multiple transcription start sites (Maue et al., Neuron,4:223, 1990; and Sauerwald et al., J Biol Chem, 265:14932, 1990). Thenucleotide sequence, between the human and chick promoter regions, waspoorly conserved. However, there are consensus transcription factorbinding sites located in similar positions in the two promoters(Matter-Sadzinski et al., supra, 1992). These include SP-1 and AP-2binding sites. SP-1 and AP-2 consensus motifs are frequently found inother ligand-gated ion channel genes (See e.g., Bessis et al., NuclAcids Res, 21:2185, 1993), and may contribute to neuronal specificity.

A cyclic AMP response element (CREB) binding site motif was alsoidentified in the human promoter, but is not found in the chick gene.The presence of this CREB site in the human promoter is interestingsince the mammalian α7 gene is known to be down-regulated bycorticosterone (Pauly et al., “Glucocorticoid Regulation of Sensitivityto Nicotine,” in The Biology of Nicotine: Current Research Issues, RavenPress, New York, N.Y., pp. 121-139, 1992), which affects expression ofthe CREB-binding protein. Thus, it is contemplated that corticosteroneand other glucocorticoids affect the α7 gene in some embodiments of thepresent invention.

In addition, alternative splicing of the full-length α7 gene wasdetected during the development of the present invention. Six differentsplice variants were identified through sequencing full-lengthtranscripts. However, only one, missing exon 3, did not interrupt theframe of translation.

Several important motifs, which affect correct splicing of heterogeneousRNA, were identified during the development of the present invention.For example, there are two Chi(X) sequences (consensus: CCTGGTGG) knownto enhance splicing, present in the human α7 gene of the presentinvention; there is one in intron 4 and one in the 3′-UT of the cDNA.Another group of splice enhancers with sequence (T)GCATG(A), have beenlocalized as well. There are seven motifs of this enhancer class insequence identified for intron 2 (approximately >25 kb in size). Anadditional enhancer of this type has been found in the large intron 4.It is contemplated that additional splicing motifs are localized in thehuman α7 sequence.

Exons 5-10 of the α7 nicotinic receptor gene were found to be duplicatedin the human genome. The duplicated sequences lie within 1 Mb and arecentromeric to the full-length α7 gene on chromosome 15. The evidencefor the duplication includes mapping of the duplicated sequences to adifferent site on a YAC contig of the region. Additionally, heterozygouspolymorphic sequences at exonic sites and at the L76630 locus, located1.4 kb beyond the 3′ end of the coding region, were detected in both asomatic cell chromosome 15 hybrid and in a single YAC (969b11)containing both the full-length gene and duplicated α7 exons. Theapparent arrangement of the duplication is head to tail in relation tothe full-length gene.

Further complexity for the α7 gene structure was introduced when it wasdetermined that some of the RACE clones isolated during cloning of ahuman α7 cDNA contained only exons 5-10, and additional non-α7 sequences5′ of exon 5. These sequences were identical to sequences found inseveral EST clones that were located by homology screening with α7 cDNAsequence. The EST clones also contained only exons 5-10 of the α7 gene,with the previously unreported sequences again 5′ of exon 5. PCRproducts from genomic DNA and from YACs 948a10 and 953g6 revealed thatthese non-α7 sequences were present in genomic clones containing eitherthe full-length gene or the duplicated α7 sequences, and four novelexons were defined. It is contemplated that these sequences are arrangedas alternatively spliced exons, as the positions of the consensus splicejunctions between them correspond to the spliced products seen in theRACE and EST clones. These new exons were designated as “3′-α7A,” “α7B,”“α7C” and “α7D-5′.” The RACE products were variable in their inclusionof exon B, similar to the EST clones.

Partial gene duplication has been implicated in human disease (See e.g.,Hu and Worton, Hum Mutat, 1:3, 1992; Lehrman et al., Cell, 48:827, 1987;and Den-Dunnen, et al., Am J Hum Genet, 45:835, 1989). Thus, it iscontemplated that although transcription of mRNAs containing the novelexons was found to occur at levels similar to those of the full-lengthcoding region, the novel exons may be expressed only from the duplicatedα7 sequences. There is also evidence for novel exons in another gene onchromosome 15, the small nuclear riboprotein-N(SNRPN); these exons atboth the 5′- and 3′-ends of the SNRPN gene are also transcribed asalternative mRNAs. In fact, dupCHRNA7 is missing in some people, and thedeletion of the duplicated gene is more commonly observed inschizophrenics than in control subjects.

It is contemplated that the human alternative transcripts containing thenovel exons α7D, α7C, α7B, and α7A, might be translated. Thesealternatives lack the α7 signal peptide and disulfide bridge, which havebeen shown to be necessary for assembly of the homologous alpha subunitin muscle (Blount and Merlie, J Cell Biol, 111:2613, 1990). However, analternatively spliced transcript of the muscle alpha, containing anadditional exon, is expressed at equal levels to the correctly splicedisoform. It is also translated, but not assembled and is localized tothe endoplasmic reticulum (Beeson et al., EMBO J, 9:2101, 1990; andNewland et al., J Physiol, 489:767, 1995). It is contemplated that asimilar localization occurs for the human α7 alternative transcripts,containing the novel exons, if translated. However, it is not intendedthat the present invention be limited to any particular localization ofthese alternative transcripts.

Antibodies to the cytoplasmic loop of the chick α7, between membranespanning regions III and IV, have been shown to detect α7 protein inpyramidal cells of rat hippocampus (Dominguez del Toro et al., J CompNeurol, 349:325, 1994). However, during the development of the presentinvention, no α-bungarotoxin binding (i.e., indicative of a functionalreceptor), was observed on the plasma membranes of these cells. Sinceprotein, translated from alternative α7 mRNAs containing D-C-B-A-5-10,would have the epitope used as antigen for cytoplasmic loop antibodies,it is possible that sequestered, but dysfunctional α7 protein internallylocalized would be detected as well as cell surface protein. Theabundance of the D-C-B-A-5-10 alternative transcripts, thus, raises thepossibility that they are regulatory for functional expression of α7nicotinic receptors.

Although the mechanism responsible for the gene duplication is unclear,and an understanding of the mechanism is not necessary in order to usethe present invention, two alu repeats were found in the genomic clones.One is located in intron 4, 500 bp upstream of exon 5. The second islocated in the 3′-end of the gene outside of the poly-adenylation site.Alu repeats are known to have several possible functions, including aseither positive or negative enhancers of transcription. In addition,they have also been shown to mediate duplication or deletion of DNAsequences (Schmid, Prog Nucl Acid Res, 53:283, 1996; and Lehrman et al.,Cell, 48:827, 1987).

It is clear that the duplicated and expressed sequences involving thehuman α7 nicotinic receptor gene of the present invention provide themethods and compositions needed for mutation screening in disease. Thepresent invention also provides methods and compositions for treatment(including, but not limited to gene therapy) of deficits in α7expression and/or function.

The present invention provides methods and compositions needed todetermine the control of α7 expression, through the use of the DNAsequences in its promoter region, as well as DNA sequences located atits intron/exon boundaries, and DNA sequences present elsewhere in itsintrons. In addition, the present invention provides the locations andsequences of newly identified duplicated and additional exons. It iscontemplated that these sequences may be involved in pathogenicmutational events. Although the coding sequence of α7 shares somesimilarities between various animals (e.g., chickens, rodents, andhumans), the genomic structure provided in the present invention in thepromoter and introns is unique to humans, and could not have beenpredicted based on the knowledge of the genome structure of any otherspecies.

Furthermore, the coding region alone cannot be used for geneticscreening of individuals to identify mutations, because the appropriateprimers (e.g., for PCR) are needed from introns positioned outside ofthe coding region. In addition, the genomic sequence is necessary forthe production of cell lines and transgenic animals (i.e., for modelsuseful for the development of therapeutic targets in drug discovery).The present invention provides the needed genomic sequences and primersfor genetic screening methods and drug discovery.

III. Association of CHRNA7 Promoter Variants with P50 InhibitoryDeficits

Schizophrenia is a complex disorder, in which heterogeneity, reducedpenetrance, and environmental factors have made identification ofgenetic defects difficult. The work of many investigators has resultedin the discovery and replication of eight principal linkage regions inthe human genome. These include linkages at chromosome 1q21-q22(Brzustowicz et al., Science, 288:678-682, 2000), chromosome 6p22-p24(Straub et al., Mol Psychiatry, 1:89-92, 1996), chromosome 6q21-q22 (Caoet al., Genomics, 43:1-8, 1997), chromosome 8p21-p22 (Blouin et al., NatGenet, 20:70-73, 1998), chromosome 10p11-p15 (Faraone et al., Am J MedGenet, 81:290-295, 1998), chromosome 13q14-q32 (Blouin et al., supra,1998), chromosome 15q13-q15 (Riley et al., Am J Med Genet, 96:196-201,2000; Stober et al., Am J Med Genet, 67:1201-1207, 2000, Stassen et al.,Am J Med Genet, 96:173-177, 2000; Freedman et al., Am J Med Genet,105:20-22, 2001; Liu et al., Am J Med Genet, 105:658-661, 2001; Xu etal., Am J Med Genet, 105:669-674, 2001; Tsuang et al., Am J Med Genet,105:662-668, 2001; and Gejman et al., Am Med Genet, 105:789-793, 2001),and chromosome 22q11-q13 (Pulver et al., Am J Med Genet, 54:36-43,1994). Additional linkages on six other chromosomes may be contributoryin some populations (Baron, Am J Med Genet, 68:299-312, 2001). Ingeneral, linkage in any given cohort is found in only a subset of thetotal number of families examined, indicating that abnormalities indifferent gene sets may result in the same illness. Identification ofpathogenic mutations in candidate genes that lie in the major linkageregions is necessary for a rigorous understanding of how several genesinteract in the development of schizophrenia. As is described in moredetail in Examples 11-16, the inventors provide evidence that functionalpolymorphisms in the promoter region of the α7 neuronal nicotinicacetylcholine receptor subunit gene (CHRNA7 or α7), a candidate gene inthe 15q13-q14 linkage region, were more frequently found inschizophrenic patients and were associated with a sensory deficit foundin this common mental illness.

The CHRNA7 gene cluster maps to a region of replicated linkage inschizophrenia on chromosome 15q13-q14 (See, FIG. 11). D15S1360, apolymorphic marker in intron 2 of the CHRNA7 gene, is genetically linkedto a sensory deficit trait in the disease, namely a failure to inhibitthe response to repeated auditory stimuli in the immediate environment(lod=5.3, θ=0.0, P<0.001 as described Freedman et al., Proc Natl AcadSci USA, 94:587-592, 1997). Linkage to schizophrenia was also positivein this study of nine families, although not as significant (lod=1.33).Additional evidence for linkage of this locus to schizophrenia as thephenotype was found in pedigrees from the National Institute of MentalHealth (NIMH) Schizophrenia Genetics Initiative (Freedman et al., Am JMed Genet, 105:20-22, 2001; and Leonard et al., Am J Med Genet,81:308-312, 1998). A sibpair analysis showed that a significantproportion of D15S1360 alleles were shared identical-by-descent in theschizophrenics (0.58; P<0.0024). In a transmission disequilibrium studyof schizophrenia, significant genotype-wise disequilibrium (P<007) wasfound at D15S165, a polymorphic simple sequence marker localized withinone megabase of the α7 nicotinic receptor gene at 15q13-q14 (Freedmen etal., Am J Med Genet, 105:20-22, 2001). Recently a full genomic linkageanalysis was completed of the NIMH Schizophrenia Initiative pedigrees,for which the genotyping was available from Millenium Pharmaceuticals(Cambridge, Mass.). A parametric genetic analysis and an autosomalcodominant model was used, with a diagnosis of schizophrenia andschizoaffective disorder, depressed type, as the affected phenotype. Onegenetic linkage was found significant by genome-wide criteria(multipoint lod score, 3.94; P=0.00005), to the locus on 15q13-q14,within one cM of the previous finding for linkage to the locus of the α7nicotinic receptor gene (Freedman and Leonard, Schizophr Res, 49:70,2001; and Freedman et al., Am J Med Genet, 105:794-800, 2001. Severaldifferent groups have independently replicated this finding by usingnonparametric methods in the NIMH sample (Kaufmann et al., Am J MedGenet, 81:282-289, 1998), and in other samples. The same region has beenlinked to juvenile myoclonic epilepsy (Elmslie et al., Hum Mol Genet,6:1329-1334, 1997) and more recently to bipolar disorder (Edenberg etal., Am J Med Gen, 74:238-246, 1997; and Turecki et al., Mol Psychiatry,6:570-578, 2001), indicating that the locus may contain defects in agene or genes common to several neuronal disorders.

Biological and pharmacologic evidence also supports the CHRNA7 gene as acandidate gene for schizophrenia (Adler et al., Schizophr Bull,24:189-202, 1998; and Leonard et al., “The role of nicotine andnicotinic receptors in psychopathology,” in Arneric and Brioni (eds.)Neuronal Nicotinic Receptors: Pharmacology and TherapeuticOpportunities. New York, N.Y.: Wiley-Liss Inc., pp 305-320, 1999).Nicotine normalizes a sensory gating abnormality, the P50 inhibitorydeficit, found in most patients with schizophrenia and in 50% of theirfirst-degree relatives (Adler et al., Biol Psychiatry, 32:607-616, 1992;Adler et al., Am J Psychiatry, 150:1856-1861, 1993; and Freedman et al.,Schizophr Res, 4:233-243, 1991). This trait, which involves inhibitionof the response to repeated stimuli presented through the auditorysystem to the brain, can be measured by means of auditory evokedpotentials in a paired pulse paradigm. Electrodes on the scalp recordwaves with a 50 millisecond latency (P50) following paired auditorystimuli delivered 0.5 second apart (Freedman et al., Schizophr Res,4:233-243, 1991; and Baker et al., Biol Psychiatry, 22:603-617, 1987).In a normal response, the subject decreases the amplitude of the secondresponse (test response), compared with the response to the firststimulus (conditioning response), through the action of an inhibitoryneuronal pathway. The results are reported as the P50 test-conditioning(T/C) ratio. More than 85% of schizophrenic patients have abnormallyincreased P50 ratios, where the test response is greater than expectedin the normal population (Adler et al., Biol Psychiatry, 17:639-654,1982; Clementz et al., Schizophr Res, 30:71-80, 1998; Yee et al., JAbnorm Psychol, 107:691-698, 1998; Erwin et al., Schizophr Res,33:157-167, 1998; and Patterson et al., Arch Gen Psychiatry, 57:57-64,2000). This P50 inhibitory deficit is inherited in families ofschizophrenic patients in an apparently autosomal dominant pattern(Freedman et al., Proc Natl Acad Sci USA, 94:587-592, 1997; Freedman etal., Somat Cell Mol Genet, 13:479-484, 1987; and Clementz et al., Am JPsychiatry, 155:1691-1694, 1998). Thus, half of family members haveaberrant gating of the P50 auditory evoked potential, whether or notthey have the disease. The increased incidence in schizophrenic patientsand their families indicates that the P50 deficit represents a traitthat predisposes to schizophrenia. The deficit is also present, but atmuch lower levels, in the general population, in subjects with nofamilial history of schizophrenia (Waldo et al., Biol Psychiatry,47:231-239, 2000). The P50 inhibitory deficit, as previously discussed,is also genetically linked to 15q13-q14 (Freedman et al., Proc Natl AcadSci USA, 94:587-592, 1997; and Coon et al., Biol Psychiatry, 34:277-289,1993).

It is contemplated that the deficit in P50 inhibition, reflectsdecreased activity or expression of the CHRNA7 receptor. Pharmacologicantagonists of the CHRNA7 receptor reproduce the inhibitory deficit inseveral animal models (Luntz-Leybman et al., Brain Res, 587:130-136,1992; and Rollins et al., Soc Neurosci Abstr, 19:837, 1993). The DBA/2jmouse strain has 50% lower levels of CHRNA7 than most other inbredstrains, it does not show inhibition of its auditory evoked response torepeated stimuli, and the inhibition is normalized by both nicotine anda specific agonist of the α7 receptor, 2,4-dimethoxybenzylideneanabaseine (Stevens et al., Neuropsychopharm, 15:152-162, 1996; andStevens et al., Psychopharmacology, 136:320-327, 1998). The inventorshave previously found that the expression of the CHRNA7 gene was alsodecreased by approximately 50% in human postmortem hippocampus isolatedfrom schizophrenic subjects, compared with matched control subjects(Freedman et al., Biol Psychiatry, 38:22-33, 1995). Expression of theCHRNA7 gene was also decreased in different brain regions, including thereticular thalamic nucleus and frontal cortex, in schizophrenic subjects(Court et al., J Neurochem, 73:15980-1597, 1999; and Guan et al.,Neuroreport, 10:1779-1782, 1999).

As described in more detail in Examples 1-10, a genomic clone for thehuman CHRNA7 subunit was isolated from a yeast artificial chromosome(YAC) library. Mapping of the gene showed that exons 5 to 10 of theCHRNA7 gene were duplicated as part of a large DNA cassette. Theduplication was inserted approximately one Mb proximal to thefull-length α7 gene and directly 3′ of five novel exons (D′-D-C-B-A).The duplicated exons 5 to 10 are expressed with the novel exons D′-A(dupCHRNA7) as messenger RNA in both human brain and peripheral tissues.Interestingly, dupCHRNA7 was homozygotically missing in five (4.2%) of118 schizophrenic patients, but not in 59 control subjects. Mutationscreening of the amino acid coding region for the full-length CHRNA7 anddupCHRNA7 genes, and a core promoter region for the full-length gene,has been completed during development of the present invention. Althoughmultiple polymorphisms were found in the coding region, almost all weresynonymous.

In addition, a core promoter region for the full-length CHRNA7 gene wasisolated that supports efficient transcription of the reporter gene,luciferase. This 231 base pair fragment contains consensus binding sitesfor a number of transcription factors, including stimulating proteinSp1, activator protein AP-4, and a corticosteroid-responsive element,SRE as determined by using MatInspector (Quandt et al., Nucleic AcidsRes, 23:4878-4884, 1995). The regions near the Sp1 binding sites containseveral G/C-rich areas, which are contemplated to be binding sites forother transcription factors, such as Egr1. The location and spacing ofthese sites with respect to the start of exon 1 are conserved in thebovine α7 gene, where they have been shown to regulate transcription(Carrasco-Serrano et al., J Biol Chem, 273:20021-20028, 1998). Duringdevelopment of the present invention, mutation screening of thisfragment in human DNA samples from control and schizophrenic subjects,showed a large cluster of polymorphisms, many lying in these putativetranscription factor binding sites.

Although schizophrenia has a large genetic component, it is thought tobe oligogenic (Freedman et al., Am J Med Genet, 105:794-800, 2001; andGershon, Biol Psychiatry, 47:240-244, 2000). Heterogeneity in theinheritance of predisposing traits further complicates the orderlyprocess of gene identification. At present, there are 14 chromosomes onwhich genetic linkage to schizophrenia has been identified or issuggested (Baron, Am J Hum Genet, 68:299-312, 2001). Many of theseregions are contemplated to contain a gene variant contributing to thedisease in the linked populations, indicating that many genes mayinteract in the disorder, but that not all the gene variants at theseloci may be present in a single individual. Furthermore, the actualpolymorphism present in any given gene is contemplated to result indifferences in gene expression between subjects, which can also beaffected by other genes and environmental factors. Some variants arecontemplated to manifest in early development and some during puberty orpostpuberty, when schizophrenia is usually first diagnosed.Additionally, some gene variants are contemplated to compensate forothers, or to actually have a beneficial effect.

Three principal issues contribute to a discussion of the presentinvention. First, the study of a candidate gene for an endophenotype inschizophrenia, rather than the multigenic disease itself, has permittedthe identification herein of a single gene defect. Endophenotypic traitsfound in complex disorders have been examined in attempts to simplifythe biology and genetics of schizophrenia (Venables, “Input dysfunctionin schizophrenia,” in Maher (ed.) Progress in Experimental PersonalityResearch, New York, N.Y.: Academic Press, pp. 1-47, 1964; and Freedmanet al., Biol Psychiatry, 45:551-558, 1999). Examples of such traits areinhibitory gating of the P50 auditory evoked response (Freedman et al.,Biol Psychiatry, 45:551-558, 1999; and Freedman et al., Somat Cell MolGenet, 13:479-484, 1987), and smooth-pursuit eye tracking (Holzman, IntRev Neurobiol, 27:179-205, 1985; and Holzman et al., Arch GenPsychiatry, 45:641-647, 1988), both of which are found in the generalpopulation at lower levels than in the disease. In control subjects withno history of psychosis, variants in only one or a few different genesmay be required to produce a specific abnormal phenotype or trait. In adisease such as schizophrenia, interdependence of multipleneurotransmitters in a single brain pathway and the presence of multiplegene defects may worsen performance in a given quantitative trait.However, even in schizophrenia, only a subset of the genes involved inthe full clinical diagnosis is contemplated to be associated with aspecific endophenotype.

Second, the nicotinic acetylcholine receptor subunit gene, CHRNA7, wasimplicated as a candidate gene in the 15q13-q15 linkage region forschizophrenia by genetic and biological data, supporting its role insensory processing deficits in the disease (Leonard et al., Eur JPharmacol, 393:237-242, 2000; and Leonard et al., Restor NeurolNeurosci, 12:195-201, 1998). Expression of the CHRNA7 gene is decreasedin postmortem brain isolated from schizophrenic subjects compared withthat of controls (Freedman et al., Biol Psychiatry, 38:22-33, 1995;Court et al., J Neurochem, 73:1590-1597, 1999; and Guan et al., JNeuroreport, 10:1779-1782, 1999). However, the present invention is thefirst description of CHRNA7 alleles associated with decreased α7expression. Specifically, the promoter variants in CHRNA7 identifiedherein are expected to contribute to the decreased expression of thisgene in vivo. As described in Example 15 below, several of thepolymorphisms have been tested in an in vitro reporter gene assay, where6 of 8 variants were found to have decreased transcriptional activity.In fact, the most common variant at −86 bp, associated withschizophrenia (P=0.04), decreased transcription of the luciferasereporter gene by 20% (P=0.0001). Comparable transcriptional effects havebeen seen for other gene promoters (e.g., presenilin 1, tumor necrosisfactor, and paraoxonase) with single-base pair mutations (Theuns et al.,Hum Mol Genet, 9:325-331, 2000; Knight et al., Nat Genet, 22:145-150,1999; and Brophy et al., Am J Hum Genet, 68:1428-1436, 2001). Many ofthe single and double promoter variants identified herein, were foundprincipally in schizophrenic patients. Indeed, the functional variantsisolated thus far are statistically more prevalent in schizophrenicsubjects (P=0.007) than in controls. Additionally, the double variantsexamined thus far, where more than one variant was present, werecombinations of the known single variants, and were found on separatealleles. This indicates inheritance of one mutation from each parent.

It is possible that some variants in the core promoter region have beenmissed because of ascertainment bias. The sample studied herein includedmore schizophrenic subjects than controls, and had fewer AfricanAmericans and Hispanic subjects than whites. Polymorphisms at −92 bp,−143 bp, −180 bp, and −241 bp were found more often in schizophrenicpatients, but were rare in the study sample. Thus, when additionalAfrican Americans, Hispanics, and other ethnic cohorts are screened,more subjects with these rare variants (and possibly even new variants)are expected to be identified. Furthermore, during development of thepresent invention, an additional 2302 bp of sequence upstream of theCHRNA7 core promoter was isolated. Preliminary analysis of two subclonesindicated the presence of upstream repressor elements. Upstreamregulatory elements have been found in several other nicotinic receptorsubunit genes indicative of complex developmental and tissue-specificregulation of expression (Flora et al., Eur J Pharmacol, 393:85-95,2000; and Melnikova et al., Eur J Pharmacol, 393:75-83, 2000). Otherfunctional or more complex variants in schizophrenic subjects arecontemplated to lie in these regulatory regions of the human α7nicotinic receptor subunit gene, perhaps in disequilibrium with some ofthe polymorphisms in the core promoter. Although the frequency of corepromoter polymorphisms in multiply affected families was small, thepolymorphisms associated with decreased α7 expression are expected tocontribute to the transmission of sensory processing deficits inschizophrenia.

Third, because the CHRNA7 gene was targeted as a candidate gene ashaving a biological role in a sensory processing endophenotype seen inmost schizophrenic patients and in one half of their first degreerelatives (Leonard et al., “The role of nicotine and nicotinic receptorsin psychopathology,” in Arneric and Brioni (eds.), Neuronal NicotinicReceptors: Pharmacology and Therapeutic Opportunities, New York, N.Y.:Wiley-Liss Inc., pp. 305-320, 1998; and Leonard et al., Restro NeurolNeurosci, 12:195-201, 1988), it is significant that a measure ofauditory evoked inhibition in humans (the P50 gating phenotype), iscorrelated with the presence or absence of variants in the CHRNA7 corepromoter. Inhibition of the P50 response is abnormal in mostschizophrenic patients, where the test response is often larger than theconditioning response, resulting in T/C ratios much greater than 0.50.In control subjects with no history of schizophrenia, a T/C ratio rangelower than in schizophrenic patients was found (t₂₀₅=8.49, P<0.0001).However, the ratios were significantly higher in controls with promotervariants than in controls with no polymorphisms (P=0.0001). Therelationship between the presence of a promoter polymorphism and the P50T/C ratio appeared to place the control subjects into three groups. Thegrouping is contemplated to indicate either a gene dosage effect or thepresence of additional gene interactions. Inhibitory pathways inschizophrenic subjects are contemplated to be much more complex than inindividuals with no history of mental illness. Measurement of the P50phenotype in control subjects is, thus, contemplated to be lesscomplicated and more representative of the effect of a few genes orpossibly even a single gene defect. The present results indicate thatthe α7 promoter variants are associated with a measurable phenotypefound in the general population, but present more frequently inschizophrenia. Coincidentally, other investigators have notedcorrelations between higher P50 ratios and schizotypy (Croft et al.,Biol Psychiatry, 50:441-446, 2001), particularly in individuals with afamily history of schizophrenia (Cadenhead et al., Am J Psychiatry,157:1660-1668, 2000). However, none of these investigators hadidentified or suggested a correlation between CHRNA7 promoter variants,elevated P50 ratios, and predisposition to schizophrenia.

Last, the design and interpretation of candidate gene associationstudies, such as the present report, are not obvious. In the humanlipoprotein lipase gene, for example, it has been found that the averageindividual is heterozygous at 17 sites, probably because of acombination of historical population founding, stratification ofpolymorphic changes, and recombination (Clark et al., Am J Hum Genet,63:595-612, 1998). Not all of these polymorphisms will be functional,although they may be in disequilibrium with other variants and/or withthe disease. This emphasizes the importance of a thorough functionalanalysis of any polymorphisms associated with schizophrenia.Furthermore, the complexity and dependence on the interactions offunctional variants contributing to a complex major mental illness isconsistent with the hypothesis that many of these functionalpolymorphisms are likely to be common in the general population (Landerand Schork, Science, 265:2037-2048, 1990; and Gershon, Biol Psychiatry,47:240-244, 2000). In that regard, a variant in the catecholO-methyltransferase gene (COMT), found in 50% of non-mentally illsubjects, has recently been associated with prefrontal cortical deficitsin schizophrenia, but estimated to contribute only a small percentage ofthe risk for the disease (Egan et al., Proc Natl Acad Sci USA,98:6917-6922, 2001). Likewise, functional variants in the CHRNA7 genepromoter were found in 28% of the control subjects with no familyhistory of schizophrenia, but were strongly associated (P=0.0001) withhaving a deficit in auditory sensory processing. The genotype relativerisk for schizophrenia at one of the polymorphisms, −86 bp, was 2.39(95% confidence interval, 1.07-5.32), indicating a small but realcontribution to the disorder. This sort of inheritance of gene variantsis contemplated to be the case for many complex disorders. Indeed, arole for calpain 10 in type-2 diabetes has been recently reported, wherethe aberrant allele was found in 75% of the control population but in80% of those with diabetes (Horikawa et al., Nat Genet, 26:163-175,2000). Thus, the assemblage of a group of functional variants in oneindividual is contemplated to be required for the development of acomplex disease such as schizophrenia.

IV. Polymorphisms in CHRNA7 and dupCHRNA7

Evidence for genetic linkage to schizophrenia in the 15q13-q14 regionhas grown as marker density on the human genomic map has improved asdescribed herein and as subsequently published (Stober et al., Am J HumGen, 67:1201-1207, 2000; Riley et al., Am J Med Gen, 96:196-201, 2000;Liu et al., Am J Med Gen, 105:658-661, 2001; Tsuang et al., Am J MedGen, 105:662-668, 2001; and Xu et al., Am J Med Gen, 105:6698-674,2001). The region has also been linked to bipolar disorder (Turecki etal., Mol. Psych, 6:570-578, 2001).

A candidate gene in this region, the α7 nicotinic acetylcholine receptorsubunit gene CHRNA7, has been identified pharmacologically, as playing arole in an aberrant inhibitory pathway found in schizophrenia, the P50auditory evoked potential deficit as described herein and elsewhere(Luntz-Lebman et al., Brain Res, 587:130-136, 1992; Stevens et al.,Psychopharm, 136:320-327, 1998; Leonard et al., Pharmacol Biochem Behav,70:561-570, 2001; and Leonard et al., Eur J Pharmacol, 393:237-242,2000). The P50 deficit, an endophenotype of schizophrenia, isgenetically linked to D15S1360, a dinucleotide marker in intron 2 ofCHRNA7 (Freedman et al., Proc Natl Acad Sci USA, 94:587-592, 1997).

A. Proximal Promoter Polymorphisms

Functional gene variants have been isolated in the proximal promoterregion of CHRNA7 that appear to be associated with both schizophreniaand with the P50 deficit as described in Section III herein. Nowpolymorphisms in the coding region and intron/exon borders of the CHRNA7gene cluster in schizophrenic and control subjects are presented.

The mutation screening was complex, due to the partial duplication ofthe α7 gene. Exons 5-10, and intervening introns, were duplicated andinserted with a large cassette of DNA into a position proximal to thefull-length CHRNA7. The duplicated exons are expressed as mRNA with fivenon-α7 exons in several tissue types, including postmortem brain(dupCHRNA7, See, GenBank Accession No. AF029838). Thus, mapping wasrequired, for polymorphisms found in exons 5-10 in genomic DNA, toeither the full-length CHRNA7 gene or the dupCHRNA7 gene. Transcriptswere isolated, specific for each gene, from either postmortem braintissue or lymphoblasts.

Variants in both coding region and introns were identified. As shown inTable 17, 21 polymorphisms were found the exons, nine of which changedan amino acid. Three of these amino acid changes, although rare, mappedto the full-length gene. These three amino acids are conserved betweenhuman, mouse (GenBank accession #A57175) and rat (GenBank accession#T01378) genes. One amino acid change in exon 4 (1112V) lies in part ofthe putative agonist-binding site (Galzi et al., Annu Rev Pharmacol,31:37-72, 1991). In the three families, in which these amino acidchanges occurred, cosegregation with neither the P50 deficit nor withschizophrenia was observed. In such a complex disorder, bilinealinheritance or reduced penetrance is contemplated to explain thisresult. However, functional promoter variants were found in all three ofthese families as described herein and as published (Leonard et al.,Arch Gen Psychiatry, 59:1085-1096, 2002).

Ten intronic variants and two variants in the 3′-untranslated regionwere identified. Two polymorphisms in introns 2 and 3 were in thefull-length gene, but the seven variants in introns 7 and 9 and those inthe 3′untranslated region (3′UTR) could not be easily mapped because ofthe gene duplication. One variant in intron 9 at +37 was associated withschizophrenia in African Americans (X²=9.986, 1; P=0.0016) and was inlinkage disequilibrium with a synonymous variant mapped to CHRNA7. Anumber of the intronic polymorphisms either introduce a cryptic splicesite or alter a splice site. The 2 bp deletion at 497/8, present in theduplicated gene in more than 50% of subjects examined, disrupts anexonic splice enhancer site (EXE). Thus, if exon 6 were aberrantlyspliced out in this gene variant, the translation of a putative proteinwould remain in frame, indicating that this splice variant hasregulatory effects. Interestingly, multiple alternatively splicedtranscripts were identified in initial studies of the α7 gene cluster asdescribed in herein. Splice variants have been found to be a commoncausal element in disease (Ars et al., Hum Mol Gen, 9:237-247, 2000;Grabowski and Black, Prog Neurobiol, 65:289-308, 2001; and Cartegni etal., Nat Rev Gen, 3:285-298, 2002). Since the CHRNA7 receptor assemblesas a pentamer, the presence of splice variants represents a possiblemechanism for dominant-negative decreased expression (Garcia-Guzman etal., Eur J Neurosci, 7:647-655, 1995).

The partial duplication of exons 5-10 and flanking regions not onlyintroduced complexity into the mutation screen, but suggests yet anothermechanism of mutation. The duplicon containing α7 exons 5-10 wasinserted 3′ of five exons, duplicated from another gene, and the chimerais transcribed in both lymphocytes and brain. This fusion gene or geneproduct is contemplated to interfere with expression, assembly orfunction of the CHRNA7 gene product in a manner similar to a splicevariant. Variants in transcribed regions, common to both the CHRNA7 anddupCHRNA7 genes were mapped in mRNA from only a limited number ofindividuals. Thus, it is also contemplated that gene conversion plays arole in disruption of full-length CHRNA7 in some individuals.

Further, presence of the partial duplication is contemplated to lead todeletion or additional duplication events. For instance, the duplicatedsequence is contemplated to prime misalignment, then recombination andsubsequent deletion of the intervening sequences including part of thefull-length gene. Deletions primed by duplications have been extensivelycharacterized in Prader Willi and Angelman syndromes, which map nearbyat 15q11-q13 (Robinson et al., J Med Gen, 35:130-136, 1998). In thisregard, five schizophrenic subjects with homozygotic deletions of theduplicated gene have been identified, although none of these subjectsappears to be missing any part of the full-length gene. Deletion of bothcopies of dupCHRNA7 has not yet been observed in controls.

Although a large number of polymorphisms were found in both thefull-length CHRNA7 gene and its partial duplication, no nucleotidechanges that either cosegregate with the P50 gating deficit orschizophrenia, or that obviously disrupt the function of the full-lengthCHRNA7 gene were isolated. In addition, none of the coding regionvariants were found to be in linkage disequilibrium with a functionalpromoter mutation. Previously, a decreased expression of CHRNA7receptors in several regions of postmortem brain in individuals withschizophrenia compared to control subjects has been observed. Since noprominent coding region mutations were found, the promoter polymorphismsdescribed herein in Section III are contemplated to be particularlyimportant, as are intronic variants in the gene. These results alsoindicate that α7 nicotinic receptors in schizophrenic subjects, thoughreduced in number, are functionally normal and thus are contemplated torespond to therapies that modulate α7 activity or response.

B. Distal Promoter Polymorphisms

The data presented herein utilize overlapping genomic fragments toidentify at least thirty-five (35) SNPs in the 2 kb 5′ upstreamregulatory region of the CHRNA7 gene. SNPs were genotyped utilizing acombination of heteroduplex analysis by denaturing high-performanceliquid chromatography and sequencing, and were analyzed for associationwith schizophrenia in Caucasian-Non Hispanic and African-Americansubjects. Smoking history was considered as a secondary outcome. Theresults show significant association of a specific SNP, rs3087454 (−1831bp), in the 5′ upstream regulatory region of the CHRNA7 gene withschizophrenia.

In one embodiment, the present invention contemplates detecting distalα7 promoter polymorphisms using Hardy Weinberg Equilibrium (HWE)Conformance And Linkage Disequilibrium Estimations calculated inaccordance with Example 21.

Thirty-five polymorphisms (i.e., for example, a single nucleotidepolymorphism, SNP) were identified in the 2 kb of a CHRNA7 5′-upstreamregulatory sequence (SEQ ID NO: 181). Many of these SNPs reside intranscription factor binding sites. See, FIG. 18. The minor allelefrequency (MAF) of twenty-six (26) SNPs occurring in Caucasian-NonHispanic subjects and/or in African-American subjects are presented.See, Table 23.

TABLE 23 Chromosomal location and minor allele frequencies for SNPs inthe regulatory region of CHRNA7 Chromosomal Minor allele frequency Minorallele frequency SNP marker rs # location (bp) (Caucasian-Non Hispanic)(African-American) −46 G/T 30110044 0.003 0.097 −80 G/A 30110010 0.0030.0 −86 C/T 30110004 0.064 0.004 −92 G/A 30109998 0.006 0.004 −93 C/G30109997 0.003 0.0 −178 −G 30109912 0.004 0.076 −190 +G 30109900 0.0010.05 −191 G/A 30109899 0.001 0.0 −194 G/C 30109896 0.042 0.02 −241 A/G30109849 0.001 0.0 −316 C/A 30109778 0.004 0.0 −518 C/T 30109306 0.00.017 −653 G/A 30109441 0.001 0.0 −704 C/A 30109390 0.007 0.0 −768 T/A30109326 0.003 0.003 −813 C/A 30109281 0.0 0.001 −905 T/C 30109189 0.0010.003 −929 C/G 30109165 0.0 0.002 −1247 T/A 30108843 0.001 0.0 −1248 G/C30108842 0.001 0.0 −1252 C/A 30108838 0.0 0.053 −1313 C/T rs382602930108777 0.205 0.436 −1382 G/A 30108708 0.001 0.0 −1452 G/C 301086380.001 0.0 −1512 T/G rs6494165 30108578 0.059 0.081 −1831 C/A^(a)rs3087454 30108259 0.360 0.350 ^(a)The minor alleles are different inAfrican-Americans and Caucasians at −1831 bp C/A (rs3087454). InAfrican-Americans, C is the most common allele and A is the minorallele. In Caucasians, A is the most common allele and C is the minorallele.

Genotype frequencies of all SNPs were consistent with HWE (all P>0.05;data not shown). Only SNPs with MAFs ≧0.05 in at least one ethnicpopulation were considered for statistical analyses. Of note, the minoralleles for rs3087454 (−1831 bp) were different in the African-Americanand Caucasian-Non Hispanic samples. The frequency of the rs3087454(−1831 bp) “C” allele was 0.360 in the Caucasian-Non Hispanic samples,and 0.350 for the “A” allele in the African-American samples.

A linkage disequilibrium (LD) plot for the disclosed SNPs in the corepromoter of CHRNA7 and in the 5′ upstream regulatory region of the α7gene was constructed. See, FIG. 19. LD parameter estimates weregenerated using the case-control data. Many of the found twenty-six (26)polymorphisms are rare and were not included in the LD plots. In theCaucasian sample, markers rs3087454 (−1831 bp) and rs6494165 (−1512 bp)were designated as a haplotype block using the Gabriel definition.Gabriel et al., “The structure of haplotype blocks in the human genome”Science 296:2225-2229 (2002). Two SNP marker pairs, rs3087454 (−1831bp)/rs3826029 (−1313 bp) (D′=0.89), and rs6494165 (−1512 bp)/rs3826029(−1313 bp) (D′=1.0) were in particularly strong LD as they are allcontained within a 500 bp region. See, Table 23. In the African-Americansample, SNP marker rs3826029 (−1313 bp) was in strong LD with marker−1252 bp (D′=1.0), as well as two core promoter polymorphisms (−190 bp(D′=1.0) and −46 bp (D′=1.0)). Additionally, in both sample populations,SNP markers at −905 bp/−768 bp were in LD with one another (D′=1.0) andwith a core promoter polymorphism at −191 bp (D′=1.0).

b. Association of rs3087454 (−1831 bp) with Schizophrenia

1. Case-Control Results

Eight regulatory region SNPs, −46 bp, −86 bp, −178 bp, −190 bp, −1252bp, rs3826029 (−1313 bp), rs6494165 (−1512 bp), and rs3087454 (−1831bp), were analyzed for association with schizophrenia and smoking inschizophrenia. P-values were adjusted by generating empirical P-valuesvia permutation that were corrected for the number of SNPs tested andare indicated by an (*) See, Table 24.

TABLE 24 Case-control analyses under a genotypic model of associationSchizophrenia vs Smokers vs SZ smokers vs No Diagnosis Non-Smokers SZnon-smokers SNP AA Cauc AA Cauc AA Cauc  −46 bp 0.309 0.325 0.501 0.0980.096 0.134  −86 bp 0.397 0.521 1 0.167 1 0.302  −178 bp 0.319 0.6330.50 0.575 0.618 0.372  −190 bp 0.699 0.325 0.482 0.099 0.619 0.134−1252 bp 0.639 1 0.564 1 0.601 1 rs3826029 0.864 0.733 0.288 0.209 0.6530.265 (−1313 bp) rs6494165 0.134 0.638 0.228 0.505 0.937 0.229 (−1512bp) rs3087454 0.000017, 0.0005, 0.349 0.204 0.06 0.734 (−1831 bp)0.0009* 0.013* AA = African American, Cauc = Caucasian non-Hispanic.Adjusted P-values are indicated with an (*).

A significant difference was found in the genotype distribution of thers3087454 (−1831 bp C/A) polymorphism between control and schizophrenicsubjects. In the Caucasian-Non Hispanic sample, the “C/C” genotype wasassociated with schizophrenia (P=0.0009*)(OR=1.57; 95% CI=1.31-2.19) andin the African-American sample, the “A/A” genotype was associated withschizophrenia (P=0.013*)(OR=2.0; 95% CI=1.19-3.35).

Under an allelic model, the “C” allele was only nominally associatedwith schizophrenia (P=0.005) in the Caucasian-Non Hispanic sample, andthe “A” allele was nominally associated with schizophrenia in theAfrican-American sample (P=0.005). See, Table 25.

TABLE 25 Case-Control Analysis Under An Allelic Model Of AssociationSchizophrenia vs Smokers vs SZ smokers vs No Diagnosis Non-Smokers SZnon-smokers SNP AA Cauc AA Cauc AA Cauc  −46 bp 0.331 0.326 0.519 0.0990.104 0.135  −86 bp 0.398 0.411 1 0.798 1 0.389  −178 bp 0.139 0.6340.521 0.576 0.595 0.373  −190 bp 0.695 0.326 0.342 0.69 0.610 0.135−1252 bp 0.648 1 0.573 1 0.610 1 rs3826029 0.592 0.652 0.120 0.992 0.4100.724 (−1313 bp) rs6494165 0.149 0.445 0.247 0.817 0.940 0.824 (−1512bp) rs3087454 0.005, 0.005, 0.952 0.649 0.083 0.958 (−1831 bp) 0.198*0.135* AA = African American, Cauc = Caucasian non-Hispanic. AdjustedP-values are indicated with an (*).

Polymorphism rs3087454 (−1831 bp C/A) was not significantly associatedwith the outcome of smoking status in either ethnic population despiteits high correlation with schizophrenia. P-values were combined for theethnic groups to determine the overall significance. Fisher, R. A.,“Combining independent tests of significance” Am. Stat. 2:30 (1948),See, Table 26.

TABLE 26 Combined P-values at SNP rs3087454 for case-control analysesusing Fisher's method with the outcome of schizophrenia versus controlsPolymorphism Model of association Sample P^(a) rs3087454 C/A (−1831 bp)Genotypic Controls — SZ 0.0001 Allelic Controls — SZ 0.124  ^(a)P-valueswere combined with the formula:$X_{2k}^{2} = {{- 2}{\sum\limits_{i = 1}^{k}{{\log_{e}({pi})}.}}}$Where k = degrees of freedom, and p_(i) = P-value.

As expected, this meta-analysis of the rs3087454 (−1831 bp) SNPindicated a significant association with schizophrenia (P=0.0001) undera genotypic model, but not under an allelic model (P=0.124).

The 58 childhood-onset subjects included in this study were analyzedseparately for association with rs3087454 (−1831 bp), but there was noevidence for association (P=0.560*). When childhood-onset subjects wereexcluded from the Caucasian-Non Hispanic case-control sample (a majorityof childhood-onset subjects were Caucasian), the P-value for an allelicmodel of association decreased from P=0.135* to P=0.033*. Similarly, theP— value for a genotypic model of association decreased from P=0.0009*to P=0.0002*.

In an attempt to further refine the schizophrenia phenotype, additionalanalyses were performed that included the assumption that schizophrenicsmokers may be biologically distinct from schizophrenic non-smokers.These analyses included the following dependent variables; schizophrenicsmokers versus control smokers, schizophrenic non-smokers versus controlnon-smokers, and schizophrenic smokers versus all controls. In theAfrican-American sample, nominally significant P-values at rs3087454(−1831 bp) were found with the outcomes of schizophrenic smokers versuscontrol smokers (P=0.018, genotype; P=0.508, allele), schizophrenicnon-smokers versus control non-smokers (P=0.0025, genotype; P=0.008,allele), and schizophrenic smokers versus all controls (P=0.001,genotype; P=0.606 allele). In the Caucasian-Non Hispanic sample, anominally significant P-value at rs3087454 (−1831 bp) was found with theoutcome of schizophrenic smokers versus all controls (P=0.041 genotype;P=0.179 allele).

c. Relationship of the Schizophrenia “Risk” Allele rs3087454 to RareCHRNA7 5′ Regulatory Region Polymorphisms

The frequency of a polymorphism is an important factor in determiningthe power to detect the risk allele(s) in a given association study.Many of the CHRNA7 5′ regulatory region polymorphisms are very rare withminor allele frequencies (MAF)<0.001. For example, the data was fittedto the ‘common disease-rare alleles’ model that has been suggested toexplain some cases of schizophrenia. McClellan et al., “Schizophrenia: acommon disease caused by multiple rare alleles. Br. J. Psychiatry190:194-199 (2007). The relationship between the rs3087454 (−450 1831bp) “risk” genotype and rare proximal promoter polymorphisms wasexamined. The number of individuals positive for both a proximalpromoter polymorphism and the schizophrenia “risk” genotype at rs3087454(−1831 bp) were compared against those with only proximal promoterpolymorphisms. See, Table 27.

TABLE 27 Comparison of the number of individuals positive for both 5′polymorphisms AND the schizophrenia “risk” genotype at rs3087454 againstthose with only 5′ polymorphisms and without the “risk” genotype in thecase-control sample Ethnicity Caucasian African-American Genotype atrs3087454 C/C A/C A/A A/A C/A C/C (−1831) (N = 43) (N = 193) (N = 153)(N = 7) (N = 55) (N = 41) CHRNA7 5′ Polymorphisms  −46 G/T 1 1 0 0 8 11 −86 C/T 2 20 24 0 0 0  −92 G/A 0 2 2 0 0 0  −178 −G 1 1 1 2 6 6  −190+G 0 1 0 1 8 0  −191 G/A 0 1 0 0 1 5  −194 G/C 10 22 0 0 2 1  −241 A/G 02 0 0 0 0  −316 G/A 0 1 3 0 0 0  −518 C/T 0 0 0 0 2 0  −653 G/A 0 1 0 00 0  −704 C/A 0 3 0 0 0 0  −768 T/A 0 1 0 0 1 2  −813 C/T 0 0 0 0 1 0 −905 T/C 0 1 0 0 1 3  −929 C/G 0 0 0 0 1 3 −1247 T/A 0 1 0 0 0 0 −1252C/A 0 0 0 0 3 5 rs3826029 C/T (−1313) 29 118 0 2 24 36 −1382 G/A 0 1 0 00 0 −1452 G/C 0 0 1 0 0 0 rs6494165 T/G (−1512) 11 30 0 0 3 9 The CCgenotype is associated with schizophrenia in the Caucasian samplepopulation and the AA genotype is associated with schizophrenia in theAfrican-American sample population.

Of the rare promoter SNPs in the Caucasian-Non-Hispanic samplepopulation, the SNP at −194 bp occurs more often in individuals with the“C” risk allele and the “CC” risk genotype. The D′ value for −194 bp andrs3087454 is 0.73, however, the confidence bounds are large (0.40-0.89).Of the rare SNPs in the African-American sample population, the SNP at−190 bp occurs more often in individuals with the “A” risk allele andthe “AA” risk genotype. The D′ value is 0.20 for −190 bp and rs3087454,with confidence bounds of 0.02-0.75. With many rare SNPs and relativelylow D′ values, haplotype analyses may have limited power to detect arisk haplotype due to the presence of many rare haplotypes and the errorassociated with haplotype frequency estimation. However, observationsprovided by Table 27 suggest rare CHRNA7 proximal promoter polymorphismsmay be relevant to schizophrenia as a haplotype with rs3087454 (−1831bp) or as single markers.

d. Family-Based Results

Results of the family-based PDT analysis in a second cohort supportedthe majority of results obtained in case-control studies (data notshown). At rs3087454 (−1831 bp) in the Caucasian-Non Hispanic sample,the “C” allele was found to be linked/associated with schizophrenia(nominal P=0.048). The rs3087454 SNP was not associated withschizophrenia in the African-American sample. The rs6494165 (−1512 bp)SNP was nominally associated with smoking (P=0.045) when Caucasian-NonHispanic schizophrenic non-smokers and smokers were compared, as was theproximal promoter SNP −86 bp (P=0.026). These results were notreplicated in the African-American sample.

e. Association Studies with P50 Ratios in Control Subjects

Linear regression models were estimated separately for the two ethnicgroups for the SNP at rs3087454 (−1831 bp) because different alleleswere associated with schizophrenia in African-Americans (“A” allele) andCaucasian-Non-Hispanics (“C” allele) at this biallelic marker. For allother SNPs, ethnic groups were combined in order to gain statisticalpower and ethnicity was included as a covariate. Two proximal promoterSNPs were nominally associated with higher P50 ratios in controlsubjects (SNP −86 bp, P=0.03; SNP −191 bp, P<0.01). This study did notidentify significant association with the P50 deficit at rs3087454(−1831 bp)

f. Functional Analysis of rs3087454 (−1831 bp)

SNP rs3087454 (−1831 bp) was examined for effects on α7 nicotinicreceptor gene expression in a luciferase reporter gene assay. Results offunctional studies in two cell lines (SH-SY5Y and SK-N-BE) found nosignificant difference in the functional expression of either the “A” or“C” alleles for this SNP (data not shown).

5. Discussion

The data presented herein demonstrates that polymorphisms relating toschizophrenia susceptibility is not limited to the α7 nicotinicacetylcholine receptor gene (CHRNA7) core promoter, but are also locatedin the 5′ upstream regulatory region. For example, an rs3087454 (−1831bp) polymorphism, located in a putative repressor region upstream ofCHRNA7, was significantly associated with schizophrenia in both theCaucasian-Non Hispanic and African-American case-control samples.

Nominal P-values for family-based analyses supported results in thecase-control sample. Notably, different “risk” alleles were associatedwith schizophrenia in African-Americans (“A” allele) and Caucasian-NonHispanics (“C” allele) at this biallelic marker. Associations ofdifferent alleles at the same locus with the same disease have beenreported both: i) across different ethnic groups (Singleton et al.,“alpha-Synuclein locus triplication causes Parkinson's disease” Science302:841. (2003); and Tan et al., “Alpha synuclein promoter and risk ofParkinson's disease: microsatellite and allelic size variability”Neurosci. Lett. 336:70-72 (2003)); and ii) within the same ethnic group.Park et al., “Functional catechol-O-methyltransferase gene polymorphismand susceptibility to schizophrenia” Eur. Neuropsychopharmacol.12:299-303 (2002); Glatt et al., “Association between a functionalcatechol O— methyltransferase gene polymorphism and schizophrenia:meta-analysis of case-control and family-based studies. Am. J.Psychiatry 160, 469-476 (2003); Wonodi et al., “Association betweenVal108/158 Met polymorphism of the COMT gene and schizophrenia” Am. J.Med. Genet. B Neuropsychiatr. Genet. 120:47-50. (2003); Chen et al.,“Case-control study and transmission disequilibrium test provideconsistent evidence for association between schizophrenia and geneticvariation in the 22q11 gene ZDHHC8” Hum. Mol. Genet. 13:2991-2995(2004); and Sanders et al., “Haplotypic association spanning the22q11.21 genes COMT and ARVCF with schizophrenia” Mol. Psychiatry10:353-365 (2005).

This observation could be explained by population differences whereheterogeneous effects of the same polymorphism are caused by differencesin genetic background. Alternatively, in complex diseases such asschizophrenia, in which multiple loci act jointly to confersusceptibility for disease, associations of different alleles at thesame locus could be attributed to a correlation with a causalpolymorphism at another locus. Lin et al., “No gene is an island: theflip-flop phenomenon” Am. J. Hum. Genet. 80:531-538 (2007). Thecorrelation can be due to either an interaction between the loci or tolinkage disequilibrium. Although it is not necessary to understand themechanism of an invention, it is believed that the finding thattranscription levels were not changed in an in vitro reporter geneanalysis of the rs3087454 (−1831 bp) polymorphism suggests that theresults of the presently disclosed genetic association studies arelikely due to linkage disequilibrium. Attempts to refine theschizophrenia phenotype utilizing smoking history did not improveevidence for such an association, although nominal significance wasnoted in several comparisons.

Exploratory examination of CHRNA7 proximal promoter polymorphisms andtheir relationship with rs3087454 (−1831 bp) suggests that rare CHRNA7promoter polymorphisms may occur more frequently in individuals with thers3087454 (−1831 bp) risk genotype and could be relevant toschizophrenia as a haplotype with rs3087454 (−1831 bp) or as singlemarkers in some individuals. Exclusion of the child-onset subjects inthe Caucasian case-control sample resulted in a more significant P-valuefor both allelic and genotypic models of association with schizophrenia.It is, therefore, likely that the rs3087454 “risk” allele is morerelevant to the etiology of adult onset schizophrenia.

These data show that polymorphisms in the 5′ upstream regulatory regionof the α7 nicotinic receptor gene (CHRNA7) have predictive value for theonset and development of schizophrenia. Such data suggests thatinvestigation of the CHRNA7 gene to identify the complete geneticvariance in the linkage peak region by fine mapping with a combinationof SNPs and microsatellite markers is warranted. Such studies coulduncover a causal SNP, correlated with rs3087454, and reveal whethervariance in the candidate gene CHRNA7, its upstream regulatory regions,or a relevant SNP in the partial duplication (CHRFAM7A) is associatedwith schizophrenia.

V. Detection of CHRNA7 and dupCHRNA7 Alleles

In some embodiments, the present invention includes alleles of CHRNA7and dupCHRNA7 that increase a subject's susceptibility to schizophrenia(e.g., including but not limited to alleles containing the promotervariants described herein such as −86C/T, −92G/A, −143G/A, −178−G,−180G/C, −191G/A, −194G/C, and −241A/G). Analysis of naturally occurringhuman CHRNA7 and dupCHRNA7 alleles revealed that patients with increasedsusceptibility to schizophrenia have a mutant α7 allele that results inreduced gene transcription and decreased inhibition in sensory gating(higher P50 T/C ratio). However, the present invention is not limited toCHRNA7 and dupCHRNA7 alleles with promoter polymorphisms. In fact, anyα7 polymorphism associated with schizophrenia is within the scope of thepresent invention. For example, in some embodiments, the presentinvention provides single-nucleotide polymorphisms in other regions ofCHRNA7 and dupCHRNA7 (including but not limited to those shown herein inTables 17-19).

Accordingly, the present invention provides methods for determiningwhether a patient has an increased susceptibility to schizophrenia bydetermining whether the individual has an α7 allele containing apolymorphism contributing to reduced α7 expression. In otherembodiments, the present invention provides methods for providing aprognosis of increased risk for schizophrenia to an individual based onthe presence or absence of one or more mutations in the CHRNA7 anddupCHRNA7 genes. In preferred embodiments, the mutation contributes toschizophrenia.

A number of methods are available for analysis of polymorphisms. Assaysfor detection of polymorphisms or mutations fall into severalcategories, including, but not limited to direct sequencing assays,fragment polymorphism assays, hybridization assays, and computer baseddata analysis. Protocols and commercially available kits or services forperforming multiple variations of these assays are available. In someembodiments, assays are performed in combination or in hybrid (e.g.,different reagents or technologies from several assays are combined toyield one assay). The following assays are useful in the presentinvention.

A. Direct Sequencing Assays

In some embodiments of the present invention, polymorphisms are detectedusing a direct sequencing technique. In these assays, DNA samples arefirst isolated from a subject using any suitable method. In someembodiments, the region of interest is cloned into a suitable vector andamplified by growth in a host cell (e.g., a bacteria). In otherembodiments, DNA in the region of interest is amplified using PCR.

Following amplification, DNA in the region of interest (e.g., the regioncontaining the polymorphism of interest) is sequenced using any suitablemethod, including but not limited to manual sequencing using radioactivemarker nucleotides, or automated sequencing. The results of thesequencing are displayed using any suitable method. The sequence isexamined and the presence or absence of a given polymorphism isdetermined.

B. PCR Assay

In some embodiments of the present invention, polymorphisms are detectedusing a PCR-based assay. In some embodiments, the PCR assay comprisesthe use of oligonucleotide primers to amplify CHRNA7 and/or dupCHRNA7fragment(s) containing the polymorphism of interest. The presence of anα7 allele containing nucleotide additions or deletions results in thegeneration of a longer or shorter PCR fragments respectively, which canbe detected by gel electrophoresis.

In other embodiments, the PCR assay comprises the use of oligonucleotideprimers that hybridize only to the mutant or wild type allele of α7(e.g., to the region of polymorphism). Both sets of primers are used toamplify a sample of DNA. If only the mutant primers result in a PCRproduct, then the patient has the mutant α7 allele. If only thewild-type primers result in a PCR product, then the patient has the wildtype allele of α7.

C. Fragment Length Polymorphism Assays

In some embodiments of the present invention, polymorphisms are detectedusing a fragment length polymorphism assay. In a fragment lengthpolymorphism assay, a unique DNA banding pattern based on cleaving theDNA at a series of positions is generated using an enzyme (e.g., arestriction endonuclease). DNA fragments from a sample containing apolymorphism will have a different banding pattern than wild type.

1. RFLP Assay

In some embodiments of the present invention, polymorphisms are detectedusing a restriction fragment length polymorphism assay (RFLP). Theregion of interest is first isolated using PCR. The PCR products arethen cleaved with restriction enzymes known to give a unique lengthfragment for a given polymorphism. The restriction-enzyme digested PCRproducts are separated by agarose gel electrophoresis and visualized byethidium bromide staining. The length of the fragments is compared tomolecular weight markers and fragments generated from wild-type andmutant controls.

2. CFLP Assay

In other embodiments, polymorphisms are detected using a CLEAVASEfragment length polymorphism assay (CFLP; Third Wave Technologies,Madison, Wis.; See e.g., U.S. Pat. No. 5,888,780). This assay is basedon the observation that when single strands of DNA fold on themselves,they assume higher order structures that are highly individual to theprecise sequence of the DNA molecule. These secondary structures involvepartially duplexed regions of DNA such that single stranded regions arejuxtaposed with double stranded DNA hairpins. The CLEAVASE I enzyme, isa structure-specific, thermostable nuclease that recognizes and cleavesthe junctions between these single-stranded and double-stranded regions.

The region of interest is first isolated, for example, using PCR. Then,the DNA strands are separated by heating. Next, the reactions are cooledto allow intrastrand secondary structure to form. The PCR products arethen treated with the CLEAVASE I enzyme to generate a series offragments that are unique to a given SNP or mutation. The CLEAVASEenzyme treated PCR products are separated and detected (e.g., by agarosegel electrophoresis) and visualized (e.g., by ethidium bromidestaining). The length of the fragments is compared to molecular weightmarkers and fragments generated from wild-type and mutant controls.

D. Hybridization Assays

In preferred embodiments of the present invention, polymorphisms aredetected by hybridization assay. In a hybridization assay, the presenceof absence of a given polymorphism or mutation is determined based onthe ability of the DNA from the sample to hybridize to a complementaryDNA molecule (e.g., a oligonucleotide probe). A variety of hybridizationassays using a variety of technologies for hybridization and detectionare available. A description of a selection of assays is provided below.

1. Direct Detection of Hybridization

In some embodiments, hybridization of a probe to the sequence ofinterest (e.g., polymorphism) is detected directly by visualizing abound probe (e.g., a Northern or Southern assay; See e.g., Ausabel etal. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons,NY, 1991). In these assays, genomic DNA (Southern) or RNA (Northern) isisolated from a subject. The DNA or RNA is then cleaved with a series ofrestriction enzymes that cleave infrequently in the genome and not nearany of the markers being assayed. The DNA or RNA is then separated(e.g., agarose gel electrophoresis) and transferred to a membrane. Alabeled (e.g., by incorporating a radionucleotide) probe or probesspecific for the mutation being detected is allowed to contact themembrane under a condition of low, medium, or high stringencyconditions. Unbound probe is removed and the presence of binding isdetected by visualizing the labeled probe.

2. Detection of Hybridization Using “DNA Chip” Assays

In some embodiments of the present invention, polymorphisms are detectedusing a DNA chip hybridization assay. In this assay, a series ofoligonucleotide probes are affixed to a solid support. Theoligonucleotide probes are designed to be unique to a givenpolymorphism. The DNA sample of interest is contacted with the DNA“chip” and hybridization is detected.

In some embodiments, the DNA chip assay is a GeneChip (Affymetrix, SantaClara, Calif.; See e.g., U.S. Pat. No. 6,045,996) assay. The GeneChiptechnology uses miniaturized, high-density arrays of oligonucleotideprobes affixed to a “chip.” Probe arrays are manufactured byAffymetrix's light-directed chemical synthesis process, which combinessolid-phase chemical synthesis with photolithographic fabricationtechniques employed in the semiconductor industry. Using a series ofphotolithographic masks to define chip exposure sites, followed byspecific chemical synthesis steps, the process constructs high-densityarrays of oligonucleotides, with each probe in a predefined position inthe array. Multiple probe arrays are synthesized simultaneously on alarge glass wafer. The wafers are then diced, and individual probearrays are packaged in injection-molded plastic cartridges, whichprotect them from the environment and serve as chambers forhybridization.

The nucleic acid to be analyzed is isolated, amplified by PCR, andlabeled with a fluorescent reporter group. The labeled DNA is thenincubated with the array using a fluidics station. The array is theninserted into the scanner, where patterns of hybridization are detected.The hybridization data are collected as light emitted from thefluorescent reporter groups already incorporated into the target, whichis bound to the probe array. Probes that perfectly match the targetgenerally produce stronger signals than those that have mismatches.Since the sequence and position of each probe on the array are known, bycomplementarity, the identity of the target nucleic acid applied to theprobe array can be determined.

In other embodiments, a DNA microchip containing electronically capturedprobes (Nanogen, San Diego, Calif.) is utilized (See e.g., U.S. Pat. No.6,068,818). Through the use of microelectronics, Nanogen's technologyenables the active movement and concentration of charged molecules toand from designated test sites on its semiconductor microchip. DNAcapture probes unique to a given SNP or mutation are electronicallyplaced at, or “addressed” to, specific sites on the microchip. Since DNAhas a strong negative charge, it can be electronically moved to an areaof positive charge.

First, a test site or a row of test sites on the microchip iselectronically activated with a positive charge. Next, a solutioncontaining the DNA probes is introduced onto the microchip. Thenegatively charged probes rapidly move to the positively charged sites,where they concentrate and are chemically bound to a site on themicrochip. The microchip is then washed and another solution of distinctDNA probes is added until the array of specifically bound DNA probes iscomplete.

A test sample is then analyzed for the presence of target DNA moleculesby determining which of the DNA capture probes hybridize, withcomplementary DNA in the test sample (e.g., a PCR amplified gene ofinterest). An electronic charge is also used to move and concentratetarget molecules to one or more test sites on the microchip. Theelectronic concentration of sample DNA at each test site promotes rapidhybridization of sample DNA with complementary capture probes(hybridization may occur in minutes). To remove any unbound ornonspecifically bound DNA from each site, the polarity or charge of thesite is reversed to negative, thereby forcing any unbound ornonspecifically bound DNA back into solution away from the captureprobes. A laser-based fluorescence scanner is used to detect binding,

In still further embodiments, an array technology based upon thesegregation of fluids on a flat surface (chip) by differences in surfacetension (ProtoGene, Palo Alto, Calif.) is utilized (See e.g., U.S. Pat.No. 6,001,311). Protogene's technology is based on the fact that fluidscan be segregated on a flat surface by differences in surface tensionthat have been imparted by chemical coatings. Once so segregated,oligonucleotide probes are synthesized directly on the chip by ink-jetprinting of reagents. The array with its reaction sites defined bysurface tension is mounted on a X/Y translation stage under a set offour piezoelectric nozzles, one for each of the four standard DNA bases.The translation stage moves along each of the rows of the array and theappropriate reagent is delivered to each of the reaction site. Forexample, the A amidite is delivered only to the sites where amidite A isto be coupled during that synthesis step and so on. Common reagents andwashes are delivered by flooding the entire surface, and are thenremoved by spinning.

DNA probes unique for the polymorphism of interest are affixed to thechip using Protogene's technology. The chip is then contacted with thePCR-amplified genes of interest. Following hybridization, unbound DNA isremoved and hybridization is detected using any suitable method (e.g.,by fluorescence de-quenching of an incorporated fluorescent group).

In yet other embodiments, a “bead array” is used for the detection ofpolymorphisms (Illumina, San Diego, Calif.; See e.g., PCT PublicationsWO 99/67641 and WO 00/39587, each of which is herein incorporated byreference). Illumina uses a BEAD ARRAY technology that combines fiberoptic bundles and beads that self-assemble into an array. Each fiberoptic bundle contains thousands to millions of individual fibersdepending on the diameter of the bundle. The beads are coated with anoligonucleotide specific for the detection of a given SNP or mutation.Batches of beads are combined to form a pool specific to the array. Toperform an assay, the BEAD ARRAY is contacted with a prepared subjectsample (e.g., DNA). Hybridization is detected using any suitable method.

3. Enzymatic Detection of Hybridization

In some embodiments of the present invention, genomic profiles aregenerated using a assay that detects hybridization by enzymatic cleavageof specific structures (INVADER assay, Third Wave Technologies; Seee.g., U.S. Pat. No. 6,001,567). The INVADER assay detects specific DNAand RNA sequences by using structure-specific enzymes to cleave acomplex formed by the hybridization of overlapping oligonucleotideprobes. Elevated temperature and an excess of one of the probes enablemultiple probes to be cleaved for each target sequence present withouttemperature cycling. These cleaved probes then direct cleavage of asecond labeled probe. The secondary probe oligonucleotide can be 5′-endlabeled with fluorescein that is quenched by an internal dye. Uponcleavage, the de-quenched fluorescein labeled product may be detectedusing a standard fluorescence plate reader.

The INVADER assay detects specific mutations and SNPs in unamplifiedgenomic DNA. The isolated DNA sample is contacted with the first probespecific either for a SNP/mutation or wild type sequence and allowed tohybridize. Then a secondary probe, specific to the first probe, andcontaining the fluorescein label, is hybridized and the enzyme is added.Binding is detected by using a fluorescent plate reader to compare thesignal of the test sample to known positive and negative controls.

In some embodiments, hybridization of a bound probe is detected using aTaqMan assay (PE Biosystems, Foster City, Calif.; See e.g., U.S. Pat.No. 5,962,233). The assay is performed during a PCR reaction. The TaqManassay exploits the 5′-3′ exonuclease activity of the AMPLITAQ GOLD DNApolymerase. A probe, specific for a given allele or mutation, isincluded in the PCR reaction. The probe consists of an oligonucleotidewith a 5′-reporter dye (e.g., a fluorescent dye) and a 3′-quencher dye.During PCR, if the probe is bound to its target, the 5′-3′ nucleolyticactivity of the AMPLITAQ GOLD polymerase cleaves the probe between thereporter and the quencher dye. The separation of the reporter dye fromthe quencher dye results in an increase of fluorescence. The signalaccumulates with each cycle of PCR and can be monitored with afluorimeter.

In still further embodiments, polymorphisms are detected using theSNP-IT primer extension assay (Orchid Biosciences, Princeton, N.J.; Seee.g., U.S. Pat. No. 5,952,174). SNPs are identified in this assay, byusing a specially synthesized DNA primer and a DNA polymerase, toselectively extend the DNA chain by one base at the suspected SNPlocation. DNA in the region of interest is amplified and denatured.Polymerase reactions are then performed using miniaturized systemscalled microfluidics. Detection is accomplished by adding a label to thenucleotide suspected of being at the SNP or mutation location.Incorporation of the label into the DNA can be detected by any suitablemethod (e.g., if the nucleotide contains a biotin label, detection isvia a fluorescently labeled antibody specific for biotin).

E. Mass Spectroscopy Assay

In some embodiments, a MassARRAY system (Sequenom, San Diego, Calif.) isused to detect polymorphisms (See e.g., U.S. Pat. No. 6,043,031). DNA isisolated from blood samples using standard procedures. Next, specificDNA regions containing the polymorphism of interest, about 200 basepairs in length, are amplified by PCR. The amplified fragments are thenattached by one strand to a solid surface and the non-immobilizedstrands are removed by standard denaturation and washing. The remainingimmobilized single strand then serves as a template for automatedenzymatic reactions that produce genotype specific diagnostic products.

Very small quantities of the enzymatic products, typically five to tennanoliters, are then transferred to a SpectroCHIP array for subsequentautomated analysis with the SpectroREADER mass spectrometer. Each spotis preloaded with light absorbing crystals that form a matrix with thedispensed diagnostic product. The MassARRAY system uses MALDI-TOF(Matrix Assisted Laser Desorption Ionization-Time of Flight) massspectrometry. In a process known as desorption, the matrix is hit with apulse from a laser beam. Energy from the laser beam is transferred tothe matrix and it is vaporized resulting in a small amount of thediagnostic product being expelled into a flight tube. As the diagnosticproduct is charged when an electrical field pulse is subsequentlyapplied to the tube they are launched down the flight tube towards adetector. The time between application of the electrical field pulse andcollision of the diagnostic product with the detector is referred to asthe time of flight. This is a very precise measure of the product'smolecular weight, as a molecule's mass correlates directly with time offlight with smaller molecules flying faster than larger molecules. Theentire assay is completed in less than one thousandth of a second,enabling samples to be analyzed in a total of 3-5 second includingrepetitive data collection. The SpectroTYPER software then calculates,records, compares and reports the genotypes at the rate of three secondsper sample.

F. Kits for Analyzing Risk of Schizophrenia

The present invention also provides kits for determining whether anindividual possesses an α7 allele with a specific polymorphism. In someembodiments, the kits are useful in determining whether the subject isat risk of developing schizophrenia. The diagnostic kits are produced ina variety of ways. In some embodiments, the kits contain at least onereagent for specifically detecting a mutant α7 allele. In preferredembodiments, the kits contain reagents for detecting polymorphisms inthe α7 gene promoter. In preferred embodiments, the reagents are primersfor amplifying the region of DNA containing the promoter. In otherpreferred embodiments, the reagent is a probe that binds to thepolymorphic region. In some embodiments, the kit contains instructionsfor determining whether the subject is at risk for developingschizophrenia. In preferred embodiments, the instructions specify thatrisk for developing schizophrenia is determined by detecting thepresence or absence of a mutant α7 allele in the subject, whereinsubjects having an allele containing a promoter polymorphism associatedwith decreased α7 transcription, have an increased risk of developingschizophrenia. In some embodiments, the kits include ancillary reagentssuch as buffering agents, nucleic acid stabilizing reagents, proteinstabilizing reagents, and signal producing systems (e.g., fluorescencegenerating systems). The test kit may be packaged in any suitablemanner, typically with the elements in a single container or variouscontainers as necessary along with a sheet of instructions for carryingout the test. In some embodiments, the kits also preferably include apositive control sample.

G. Bioinformatics

In some embodiments, the present invention provides methods ofdetermining an individual's risk of developing schizophrenia based onthe presence of one or more mutant alleles of α7. In some embodiments,the analysis of polymorphism data is automated. For example, in someembodiments, the present invention provides a bioinformatics researchsystem comprising a plurality of computers running a mullet-platformobject oriented programming language (See e.g., U.S. Pat. No.6,125,383). In some embodiments, one of the computers stores geneticsdata (e.g., the risk of contracting schizophrenia associated with agiven polymorphism). In some embodiments, one of the computers storesapplication programs (e.g., for analyzing transmission disequilibriumdata or determining genotype relative risks and population attributablerisks). Results are then delivered to the user (e.g., via one of thecomputers or via the internet).

VI. Treatment and Diagnosis of Schizophrenia and Other Psychoses

The present invention provides methods and compositions for thedevelopment and identification of alternative means to diagnose andtreat schizophrenia. The methods and compositions of the presentinvention will find use in the functional assessment of α7 nicotinicreceptors in schizophrenic patients, as well as for screeningpopulations for deficits in receptor function. The present inventionfinds use in genetic screening methods for genetic and parentagecounseling, as well as for identification of individuals at risk fordeveloping schizophrenia.

The present invention also provides methods and compositions formodifying α7 nicotinic receptor function. For example, the presentinvention contemplates the development of genetic therapy methods tocorrect deficiencies in the receptor structure and/or function, as wellas other therapeutic methods to enhance or decrease the function of thereceptor, as appropriate for the treatment of any given individual.

It is also contemplated that the present invention will find use inrelation to other psychosis. For example, the present invention willfind use in the diagnosis and treatment of genetic disorders, inparticular those genetic disorders known to have a genetic componentassociated with chromosome 15, such as Prader-Willi syndrome, Angelman'ssyndrome, etc., as well as other diseases, such as epilepsy (e.g.,juvenile myoclonic epilepsy), breast, and other types of cancers. Thepresent invention also finds use in the diagnosis and treatment ofnicotine-dependent illnesses, including, but not limited, to small celllung carcinoma.

Indeed, it is contemplated that the present invention will find use inthe development of antipsychotic drugs targeted to the α7 nicotinicreceptor and/or the α7 nicotinic receptor subunit gene. For example,dimethylbenzylidene anabaseine (DMXB-A; [(2-4) Dimethoxy-benzylideneanabaseine hydrochloride]) and its congeners are selectively agonisticat the α7 receptor. During the development of the present invention, ananimal model of the deficit observed in schizophrenics was used to showthat DMXB-A is effective in repeated doses, whereas the effect ofnicotine itself is completely inactivated after one dose. DMXB-A alsohas significantly less cardiovascular effects than nicotine, consistentwith its antagonist effects at α4-α2 nicotinic receptors. Thus, it iscontemplated that DMXB-A will find use as an anti-psychotic drug.

In addition to the physiological deficit found in schizophrenics andsome of their relatives, similar deficits are also found asstate-related changes in other psychotic disorders, includingParkinson's, Alzheimer's, mania and cocaine dependence. In stimulantdependence, neuroleptic anti-psychotic drugs have poor patientcompliance, possibly due to their anhedonic, catecholamine-blockingeffects. Thus, it is contemplated that nicotinic cholinergic therapeuticstrategies, such as those developed using the methods and/orcompositions of the present invention will be effective against a broadspectrum of clinical indications.

It is further contemplated that the present invention will be used todevelop antibodies and other diagnostic reagents. For example, thepresent invention finds use in the production of peptide antibodiesusing sequences identified using the present invention.

VII. Transgenic Animals

The present invention provides methods and compositions for productionof transgenic animal models of schizophrenia, nicotine-dependentillnesses, and cancer. It is also contemplated that such systems asXenopus oocytes will be used to express human α7 receptors and genesequences of the present invention.

In preferred embodiments, transgenic mice are generated usingmicroinjection of DNA containing α7 gene sequences into mammalianoocytes. However, equivalent transgenic mice can also be produced byhomologous recombination in embryonic stem (ES) cells. Techniques forthe isolation, culture, microinjection and implantation of a variety ofmammalian oocytes (e.g., murine, porcine, ovine, bovine, etc.) are knownto the art.

Two mouse models are provided in the present invention. The first modelinvolves introduction of an intact human α7 gene into the mouse genomeby microinjection of a fertilized egg with DNA from the clone containingthe full-length nAChR gene described in Example 8. The integrity of theclone in the transgenic mice is examined by PCR amplification, using allof the identified STSs on the clone map. Large flanking DNA sequencesare included in this transgene, in order to ensure proper expression ofthe human α7 gene in the mice. The expression of the human α7 gene inmice is examined by an RNase protection assay designed to specificallydetect the human α7 mRNA. This expression pattern coincides with theexpression pattern of α7 in human tissues, as analyzed by Northernhybridization. The transgenic mouse model provides animals fordeterminations of α7 function in nicotine-dependence, nicotine-dependentillnesses, cancers associated with chromosome 15, schizophrenia, andother psychoses. These animals also facilitate the development of drugsand other therapeutics that affect the function of human α7 in vivo.

The second model is exemplified using transgenic mice that containtargeted disruptions of the α7 gene. These animals, termed “knockout”animals, lack the ability to express α7 (“α7 knockouts”). In this model,mice are generated with a deletion specifically in the α7 gene, in orderto allow assessment of phenotypic changes. In order to produce thetransgenic knockout mice of the present invention, cloned human α7 genesequences are used to disrupt the α7 gene in such a manner that α7cannot be produced. In this model, two types of deletions are designed.The first removes the α7 gene entirely from the germline cells. Thesecond type of deletion is engineered so as to provide control over thespecific tissue and developmental stage in which α7 expression isinterrupted. In the second model, the viability of the mutated animalsis maintained, permitting analysis of the animals' phenotypes (includingexpression in specific tissues).

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: nAChR (nicotinic acetylcholine receptor); ° C.(degrees Centigrade); rpm (revolutions per minute); BSA (bovine serumalbumin); H₂O (water); HCl (hydrochloric acid); aa (amino acid); bp(base pair); kb or kbp (kilobase pair); Mb (megabase pair); kD(kilodaltons); gm or g (grams); μg (micrograms); mg (milligrams); ng(nanograms); μl (microliters); ml (milliliters); mm (millimeters); nm(nanometers); μm (micrometer); M (molar); mM (millimolar); M(micromolar); mM (nanomolar); pM (picomolar); U or u (units); V (volts);MW (molecular weight); sec (seconds); min(s) (minute/minutes); hr(s)(hour/hours); dNTP (deoxynucleotide); MgCl₂ (magnesium chloride); NaCl(sodium chloride); KCl (potassium chloride); DTT (dithiothreitol); DMSO(dimethyl sulfoxide); NaOH (sodium hydroxide); 3′UT (3′-untranslatedregion); OD₂₈₀ (optical density at 280 nm); OD₆₀₀ (optical density at600 nm); EST (expressed sequence tag); PAGE (polyacrylamide gelelectrophoresis); PBS (phosphate buffered saline [150 mM NaCl, 10 mMsodium phosphate buffer, pH 7.2]); PCR (polymerase chain reaction); DMEM(Dulbecco's Modified Eagle Medium); PEG (polyethylene glycol); PMSF(phenylmethylsulfonyl fluoride); RT-PCR (reverse transcription PCR); SDS(sodium dodecyl sulfate); SSC (saline-sodium citrate buffer); Tris(tris(hydroxymethyl)aminomethane); w/v (weight to volume); v/v (volumeto volume); YAC (yeast artificial chromosome); BAC (bacterial artificialchromosome); PAC (P1 artificial chromosome); RACE (Rapid Amplificationof cDNA Ends); TAFE (Transverse Alternating Field Electrophoresis); lod(maximum logarithm of the odds); STS (sequence-tagged site); Beckman(Beckman Instruments, Inc., Fullerton, Calif.); Amersham (Amersham LifeScience, Inc. Arlington Heights, Ill.); Qiagen (Qiagen Inc., SantaClarita, Calif.); Genome Systems (Genome Systems, St. Louis, Mo., USA);ICN (ICN Pharmaceuticals, Inc., Costa Mesa, Calif.); Amicon (Amicon,Inc., Beverly, Mass.); NCBI (National Center for BiotechnologyInformation, Bethesda, Md.); ATCC (American Type Culture Collection,Rockville, Md.); Research Genetics (Research Genetics, Huntsville,Ala.); Pharmacia (Pharmacia and Upjohn Diagnostics, Kalamazoo, Mich.);Boehringer-Mannheim (Boehringer-Mannheim, Indianapolis, Ind.); NationalBiosciences (National Biosciences, Inc., Plymouth Minn.); MJ Research(MJ Research, Watertown, Mass.); Perkin-Elmer (Perkin-Elmer, FosterCity, Calif.); BioRad (BioRad, Richmond, Calif.); Clontech (CLONTECHLaboratories, Palo Alto, Calif.); Gibco, GIBCO BRL, or Gibco BRL (LifeTechnologies, Inc., Gaithersburg, Md.); Gene Codes (Gene CodesCorporation, Ann Arbor, Mich.); Invitrogen (Invitrogen Corp., San Diego,Calif.); Kodak (Eastman Kodak Co., New Haven, Conn.); Promega (Promega,Corp., Madison, Wis.); New England Biolabs (New England Biolabs, Inc.,Beverly, Mass.); Novagen (Novagen, Inc., Madison, Wis.); Pharmacia(Pharmacia, Inc., Piscataway, N.J.); Schleicher & Schuell (Schleicherand Schuell, Inc., Keene, N.H.); Sigma (Sigma Chemical Co., St. Louis,Mo.); Sorvall (Sorvall Instruments, a subsidiary of DuPont Co.,Biotechnology Systems, Wilmington, Del.); Stratagene (Stratagene CloningSystems, La Jolla, Calif.); Whatman (Whatman LabSales, Hillsboro,Oreg.); Bethyl Laboratories (Bethyl Laboratories, Montgomery, Tex.);Ambion (Ambion, Inc., Austin, Tex.); and Zeiss (Carl Zeiss, Inc.,Thornwood, N.Y.).

Unless otherwise indicated, all restriction enzymes were obtained fromNew England BioLabs and were used according to the manufacturer'sinstructions.

Example 1 Samples

Samples were obtained from various normal individuals for use ascontrols in the Examples described below. To prepare these samples,blood was drawn from normal subjects, seen in the Denver SchizophreniaCenter. Of the 43 subjects used for the polymorphism analysis, 22 werefemale and 21 were male. There were 38 Caucasians, 2 Blacks, 1 Asian and2 Hispanics. None of the subjects had a history of mental illness or afamily history of mental illness.

In addition to the “normal” samples, pedigrees were selected forpresence of at least two cases of schizophrenia in a nuclear family. Twopsychiatrists made clinical diagnoses of schizophrenia, chronic type,blind to pedigree and genetic information, using Research DiagnosticCriteria (Spitzer et al., Arch Gen Psychiat, 35:773, 1978; and Endicottand Spitzer, Arch Gen Psychiat, 35:837, 1978). Nine families with 104members were studied (i.e., nine pedigrees containing individualsdiagnosed with schizophrenia were analyzed). All subjects gave writteninformed consent. Blood was also drawn from these individuals for DNAanalysis.

DNA was extracted from blood samples as described by Miller et al.(Miller et al., Nuc Acids Res, 16:1215, 1988), with one additional step.Briefly, red blood cells were lysed by incubating 10-15 ml ofanticoagulated blood at 4 C for 10 minutes in 40 ml blood cell lysissolution (BCL) (BCL contains 0.3 M sucrose, 0.01 M Tris HCl pH 7.5,0.005 M MgCl₂ and 1% Triton X-100) with occasional rocking to mix. Thecells were then centrifuged at 850×g at 4 C for 15 minutes. The pelletwas resuspended by repeated pipetting with a 1 ml wide bore glass pipetin 30 ml BCL (4° C.) and centrifuged as before.

DNA was then extracted from the pellet as described (Miller et al., NuclAcids Res, 16:1215, 1988). Briefly, the pellet was resuspended as beforein 3 ml Nuclei Lysis buffer (NL) (NL contains 0.075 M NaCl, and 0.024 MEDTA pH 8.0). Then, 200 μl of 10% SDS, 440 μl of digest diluent (1% SDS,2 mM Na₂EDTA), and 60 μl of Proteinase K (20 mg/ml stock) were thenadded to the suspension. The suspension was then incubated at 37° C. for16-20 hours with gentle mixing by inversion. Following this digestion, 1ml of saturated (approx. 6 M) NaCl was added and then the suspensionbriefly (15 seconds) was vigorously shaken. The suspension was thencentrifuged at 1340×g at room temperature for 15 minutes. Thesupernatant was transferred to a new tube, leaving the pellet at thebottom of the previous tube undisturbed. Exactly 2 volumes of absoluteethanol were added. The tube was then inverted several times until theDNA pellet was visible and floated to the top. The pellet was thentransferred to a new tube. The pellet was resuspended in 0.67 ml TE pH 8(10 mM Tris, 1 mM EDTA) by gently mixing on a roller drum for 3-5 daysat 37° C.

In addition to the samples described above, a chromosome 15 somatic cellhybrid line, R379-2B2 generously provided by Dr. Carol Jones (TheEleanor Roosevelt Institute for Cancer Research, Denver, Colo.), wasalso used. This cell line was cultured in Ham's F12, supplemented with5% fetal bovine serum and 10 μg/ml gentamicin.

Another cell line, the human neuroblastoma cell line, SH-SY5Y (Biedleret al., Cancer Res, 38:3751, 1978), was obtained from Dr. June Biedler(Memorial Sloan-Kettering Cancer Center, New York, N.Y.), and grown inDMEM/Ham's F12 (1:1 ratio, supplemented with 15% fetal bovine serum, 4mM glutamine, and 10 μg/ml gentamicin).

Example 2 Genomic Clone Isolation

In this Example, YAC clones were identified by PCR screening of twogenomic libraries, namely the St. Louis YAC library (Burke et al.,Science, 236:806, 1987) and the CEPH YAC Library 3 (Albertsen et al.,Proc Natl Acad Sci, USA, 87:4256, 1990), using α7 cDNA specific primersand methods known in the art (See e.g., Brownstein et al., Science,244:1348, 1989; Chumakov et al., Nature, 359:380, 1992; and Dracopoli etal., Current Protocols in Human Genetics, John Wiley & Sons, Inc., NewYork, N.Y., 1994).

Additional YACs, positive for loci in the α7 nAChR region wereidentified initially by using Infoclone on the CEPH/Genethon IntegratedMap courtesy of the Fondation Jean Dausset—CEPH world wide web server.Loci on the YAC contig were verified by PCR screening with either α7primer sets or primer sets for the specific polymorphic markers listedin the YAC contig (See, FIG. 6), which are available from either theCEPH database or GenBank. The PCR conditions were 94° C. for 2 minutes,1 cycle; followed by 35 cycles of 94° C. for 30 seconds, 55° C. for 30seconds, and then 72° C. for 30 seconds, followed by 72° C. for 7minutes-1 cycle. These PCR conditions were used for all PCRamplifications, unless otherwise indicated.

In addition, α7-specific primer sets were used to identify the two PACclones 64a1 and 25919. A Research Genetics BAC library was screened withα7 coding region primers by PCR to identify the BAC clone 467o18. TheBAC library purchased from Research Genetics was a “pooled DNA” library,with each hit-positive PCR product of correct size being indicative of alocation on a subsequent plate of pools. A hit on this plate gave anaddress to yet another plate, where the clone of interest was found.These “BAC clone” plates are maintained by Research Genetics. When thepositive PCR reactions produced a final plate address in the clonelibrary, that clone was ordered from Research Genetics. The PCRconditions and primers were as described herein (the primers used areshown in Tables 1 and 2). The two PAC clones (64a1 and 25919) wereidentified using the following PCR primers.

The primers used in these experiments were:

TCCTGATGTCGGCTCCCAACT (SEQ ID NO: 1) GGTACGGATGTGCCAAGGATA (SEQ ID NO:2) TTTGGGGGTGCTAATCCAGGA (SEQ ID NO: 3) TTGTTTTCCTTCCACCAGTCA (SEQ IDNO: 4) CTCGCTGCAGCTCCGGGACTCA (SEQ ID NO: 5) GGAGGCTCAGGGAGAAGTAG (SEQID NO: 6)

The first two sets of primers were used to amplify the 3′ untranslatedregion of the gene and the third primer set was used to amplify thefirst and second exons of the gene with the intervening intron 1sequence. All PCR reactions were optimized in a Perkin Elmer 480 PCRusing normal human DNA and cDNA. Conditions were as follows for thecontrol reactions in the 3′ sets: 96° C. for two min, then 35 cycles of96° C. for 30 sec, 56° C. for 30 sec, 72° C. for 1 min, and cool to 4°C., using 4 mM MgCl₂ and 10% DMSO. The 5′ PCR set was used in 1.5 mMMgCl₂ and 10% DMSO with the following conditions: 96° C. for 3 min, thencycles 1-6 were 94° C. for 1 min, 68° C.-58° C. for 1 min (dropping from68° C. to 58° C., by 2° C. increments each cycle), 72° C. for 1 min,followed by 30 cycles of 94° C. for 1 min, 58° C. for 1 min, 72° C. for1 min, then a 4 min extension at 4° C., followed by chilling at 4° C.

In these experiments, a genomic clone of the human α7 nicotinic receptorwas identified. A YAC designated as b134h10 of approximately 250 kb, wasisolated from the St. Louis YAC library. A Southern blot comparison ofYAC b134h10 with human genomic DNA indicated that it contained thefull-length α7 nAChR gene. This YAC was used to isolate a polymorphicmarker, D15S1360, as described in greater detail below.

The polymorphic marker D15S1360, a complex microsatellite with fouralleles, was isolated from a YAC containing the α7 nicotinic receptorgene. The GenBank sequence for rat α7 (GenBank Accession No. M85273) wasused to design primers to the conserved regions of the α7 codingsequence. These primers were then used to PCR amplify normal humanhippocampal cDNA obtained from a normal brain postmortem. The productswere sequenced by Automated dye-terminator chemistry (as described inExample 5). The human sequence in transmembrane regions III and IV wasthen used to design PCR primers. These primers were: 5′-CTCCAGGATCTTGGCCAAGT C-3′ (sense strand; SEQ ID NO:7), and 5′-AGATGCCCAAGTGGACCAGA G-3′ (antisense strand; SEQ ID NO:8).

The PCR reactions were conducted with 2 mM MgCl₂ and 10% DMSO, in aPerkin-Elmer 4800 using the following cycles: 94° C. for 2 min, then 5cycles of 94° C. for 1 min, 56° C. for 30 sec, 72° C. for 1 min then 35cycles 94° C. for 1 min, 54° C. for 30 sec, 72° C. for 1 min, andcooling at 4° C. The product was reamplified with primers extended tocontain a sense Xba and antisense Bam site. The products were cut andligated into a BlueScript SK-vector. Sequence of the probe was confirmedby automated dye-primer sequencing. Subsequent PCR based screening ofthe original YAC clones were based on the above primers and conditions,substituting YAC DNA for hippocampal cDNA as the template.

The PCR fragment (i.e., the probe) was sequenced and human primers weredesigned to generate a 338 bp product, which was cloned into pBluescriptSK(−). The sequence of the 338 bp probe was:

(SEQ ID NO: 9) AGATGCCCAAGTGGACCAGAGTCATCCTTCTGAACTGGTGCGCGTGGTTCCTGCGAATGAAGAGGCCCGGGGAGGACAAGGTGCGCCCGGCCTGCCAGCACAAGCAGCGGCGCTGCAGCCTGGCCAGTGTGGAGATGAGCGCCGTGGGCCCGCCGCCCGCCAGCAACGGGAACCTGCTGTACATCGGCTTCCGCGGCCTGGACGGCGTGCACTGTGTCCCGACCCCCGACTCTGGGGTAGTGTGTGGCCGCATGGCCTGCTCCCCCACGCACGATGAGCACCTCCTGCACGGCGGGCAACCCCCCGAGGGGGACCCGGACTTGGCCAAGATCCTGGA.

This probe was used to isolate a human α7 cDNA (GenBank Accession No.U40583). The Washington University human YAC library was screened withthe same primers. Two clones were isolated, B132H10 (150 kbp) andB134H10 (300 kbp), on the TAFE (Beckman) gel system, using theprocedures recommended by the manufacturer.

A sub-library of B134H10 was prepared in the λZAP phagemid vector bycomplete MboI digestion of the intact YAC DNA in a low-melt agaroseplug. The DNA was extracted and ligated into BamH1 digested andphosphatased vector, transformed into XL1Blue-(MRF′), and screened witha (CA)₁₆ (SEQ ID NO:10) oligonucleotide. One clone contained amicrosatellite [(CA)₅T(CA)₁₂TA(CA)₅C(CA)₃] (SEQ ID NO:11), which mappedto chromosome 15 (Human/Rodent Hybrid Mapping Panel #1, Coriell, CamdenN.J.). Flanking primers amplified seven additional alleles (97, 107,109, 111, 113, 115, and 117 bp). The primers used were 5′-GATCTTTGGTAGAAGC-3′ (SEQ ID NO:12), and 5′-ACCACCACTA CCATACAGAC-3′ (SEQ IDNO:13). Allele frequencies (0.006, 0.006, 0.006, 0.516, 0.370, 0.090,and 0.006; heterozygosity 0.57) were estimated from individuals marryinginto the pedigrees described in Example 1. Primer sets used for mappingα7 exons to YAC clones are listed in Table 1, below. Primers used formapping STS/dinucleotide repeat markers to YAC clones were obtained fromGenBank, and are listed in Table 2. In these Tables, and unlessotherwise indicated, all DNA sequences are shown in 5′ to 3′orientation.

TABLE 1 Primer Sets Used to Amplify Exon and Flanking Intron Sequencefrom Human Alpha-7 Nicotinic Acetylcholine Receptor Sequence AmplifiedPrimer Number Sequence SEQ ID NO: Promoter CAAAGAACGCAAGGGAGAGGT SEQ IDNO: 14 #1234 Promoter CGGCTCGCGCGCCTTTAAGGA SEQ ID NO: 15 #1235 Exon 1#1331 GGGCTCGTCACGTGGAAAAGC SEQ ID NO: 16 or #1236 Exon 1 #1233GGATCCCACGGAGGAGTGGAG SEQ ID NO: 17 Exon 2 #1198 CCTGCCCGGGTCTTCTCTCCTSEQ ID NO: 18 Exon 2 #1138 AACTAGAGTGCCCCAGCCGAGCT SEQ ID NO: 19 Exon 3#1475 AACAACGCTCTCGACAGTCAGATC SEQ ID NO: 20 Exon 3 #1476AAGATCTTGCAGCCCATGGGAG SEQ ID NO: 21 Exon 4 #1368 GGAATTCTCTTTGGTTTTGCACSEQ ID NO: 22 Exon 4 #1369 ACATATCCAGCATCTCTGTGA SEQ ID NO: 23 Exon 5#1218 TCATGCAGTCCTTTTCCTGTTTC SEQ ID NO: 24 Exon 5 #1142CTCGCTTCAGTTTTCTAACATGG SEQ ID NO: 25 Exon 6 #1124GGAACTGCTGTGTATTTTCAGC SEQ ID NO: 26 Exon 6 #1144 TTAAAGCTTGCCCAGGAATAGGSEQ ID NO: 27 Exon 7 #1143 GCTTGTGTGTGGTATACACATTG SEQ ID NO: 28 Exon 7#1126 TCCAGAGCTGATCTCAGCAGAAG SEQ ID NO: 29 Exon 8 #1125GCCCCTCGTTAGACAGAATTGAG SEQ ID NO: 30 Exon 8 #1145CTGGGCACACTCTAACCCTAACC SEQ ID NO: 31 Exon 9 #1146TGTGACGTGCAGTGCCACAGGA SEQ ID NO: 32 Exon 9 #1127AAAACCCTAGGAGGAGCCTCCTT SEQ ID NO: 33 Exon 10 #1128GATCAGCCCGTTTCCGCCTCA SEQ ID NO: 34 Exon 10 #589 GGTACGGATGTGCCAAGGATASEQ ID NO: 35 Exon A #1516 GGACTCTGCTTTTGATAAATATGT SEQ ID NO: 36 ATGExon A #1517 TTGCTGTCACTTTCTGTGTTTCAT SEQ ID NO: 37 Exon B #1283GACAATCCAAAGGTGCAGAAAGC SEQ ID NO: 38 Exon B #1538TTCGTATCTGTATACAGACAGTC SEQ ID NO: 39 Exon C #1567CCTCAGCATCATATTAGTTCAGTG SEQ ID NO: 40 Exon C #1572GCGGACAAGAGAAACAGGAAAG SEQ ID NO: 41 Exon D #1534 GGCAGTGGTGCTGTTGCCCTTSEQ ID NO: 42 Exon D #1568 TTTCTCCTGGGACTCTGGGCAC SEQ ID NO: 43

TABLE 2 STS/Dinucleotide Repeat Markers Marker GenBank Accession #D15S942 G04933 D15S1043 Z51622 D15S165 Z17271 D15S1031 Z51346 D15S1010Z53401 D15S144 Z23286 D15S1007 Z53384 D15S995 Z53051 D15S1040 Z51533

Additionally, genomic P1 artificial chromosome (PAC) clones for α7 wereobtained from Genome Systems. PAC-64-A1 is 120 kbp long and containsboth D15S1360 and the 5′ end of the coding region. L76630 was localizedin a genomic fragment containing the α7 nicotinic receptor gene(CHRNA7), isolated from a human genomic library (Stratagene), byscreening with a human α7 cDNA clone (HP411).

A 6 kbp EcoRI genomic fragment was identified, partially sequenced, andfound to include a CA dinucleotide repeat 3′ of the last exon (GenBankAccession No. L76630). Flanking primers amplified 3 alleles (180, 178,176 bp); allele frequencies were 0.06, 0.62, 0.32, with heterozygosity0.51.

PCR was performed with 1.5 mM MgCl₂: 94° C. for 5 min, 20 cycles of 94°C. for 1 min, 56° C. for 2 min, 72° C. for 1 min and 72° C. for 5 min.The two polymorphisms were genetically mapped in 96 individuals from 6reference families (Centre d'Etude du Polymorphisme Humain). Thesereference families were selected because they have three generations ofindividuals available for genotyping. Their DNA is available for geneticlocalization of markers, but their identities are confidential.

Example 3 Generation of Templates for Sequence Analysis of theIntron/Exon Borders

In this Example, extra-long PCR (XLPCR), originally described by Barnes(Barnes, Proc Natl Acad Sci USA, 91:2216, 1994), was conducted usingrTth polymerase with the Perkin Elmer XL/PCR kit (Perkin-Elmer), on aPTC 200 (MJ Research) thermal cycler with the following conditions: 94°C., 1 min, 1 cycle; 94° C., 15 sec/68° C., 10 min, 16 cycles; 94° C., 15sec/68° C., 10 min 15 sec, 12 cycles; 72° C., 10 min, 1 cycle. Enzyme,primer concentration, and dNTP concentrations were as recommended by themanufacturer. A sublibrary of YAC b134H10 was constructed by EcoRIdigestion and subcloning into Bluescript (SK−), (Stratagene), for splicejunction determination on the larger introns.

To characterize the promoter and borders around exon 1 and 2, an EcoRIand KpnI sublibrary of PAC 25919 was constructed in Bluescript (SK−). A2.9 kb clone containing exon 1, and a 5 kb clone containing exon 2 wereidentified by screening the PAC sublibrary by hybridization with an α7cDNA subclone containing 90 bp of 5′ untranslated sequence, exon 1 andexon 2.

Tentative exon borders were deduced based upon the organization of theα7 nAChR gene in the chick (Couturier et al., Neuron, 5:847, 1990).Oligonucleotide primers, as shown in the table below, were designed fromwithin the predicted exons that would amplify across the putativeintrons using extra-long PCR (XLPCR) with both genomic DNA and YACb134h10 DNA. The exon primers used were as follows. For exon 5 to exon10, the primers used were Primer #661 (TGACGCCACA TTCCACACTA A, SEQ IDNO:44); and Primer #591 (TTGTTTTCCT TCCACCAGTC A, SEQ ID NO:45). Theseprimers amplify introns 5, 6, 7, 8, and 9, with an approximate size of14 kb. For exon 3 to exon 4, the primers used were Primer #1019(CCAAGTTTTA ACCACCAACA TTTGG, SEQ ID NO:46); and Primer #1020(TCCCCGCGGA AGAATGTCTG GTTTCCAAAT CTG, SEQ ID NO:47). These primersamplify intron 3, with an approximate size of 8 kb.

The majority of intron-exon borders were determined from sequencing theXLPCR products. XLPCR products were not generated between exons 2 and 3and between exons 4 and 5, suggesting that these introns are large.Preliminary Southern blot data suggested that both are >25 kb. Theintron 2 acceptor border, and the intron 4 donor and acceptor borderswere determined after sequencing EcoRI subclones derived from YACb134h10. The intron 2 donor was determined from sequencing a KpnI/EcoRIfragment, subcloned from PAC 25919. Exon/intron border sequence andapproximate lengths for introns and exons are summarized in FIG. 1. Allof the identified intron-exon borders are consistent with 5′ donor and3′ acceptor RNA splice site consensus sequences.

The organization of the human α7 nAChR gene was found to be identical tothat found in chick with respect to number and size of exons. A signalpeptide sequence predicted by homology with the rat α7 and muscle α7coding sequences (See e.g., Séguéla et al., J Neurosci, 13:596, 1993;Conti-Tronconi et al., Proc Natl Acad Sci USA, 82:5208, 1985; and vonHeijne, Nuc Acids Res, 14:4683, 1986), was found to be encoded byexon 1. Putative glycosylation sites (See e.g., Séguéla et al., supra,1993; Schoepfer et al., Neuron, 5:35, 1990) were found in exons 2, 4 and5. The cysteine residues that form a putative disulfide bridge (Galzi etal., Ann Rev Pharmacol, 31:37, 1991), were found to be encoded by exon6. The vicinal cysteines at the acetylcholine (ACh) binding site, theα-bungarotoxin binding site, and membrane spanning region I, are allcoded by exon 7. Membrane spanning regions II and III (as in the rat)were found to be encoded by exons 8 and 9, respectively, whilemembrane-spanning region IV was found to be encoded by exon 10.

The putative promoter, and the borders for exons 1 and 2 were determinedfrom sequencing KpnI and EcoRI subclones derived from PAC 25919, whichcontains exons 1-3 and sequences 5′ of the coding region. A 2.9 kbEcoRI-KpnI fragment contained 2.6 kb of the region 5′ of exon 1, exon 1and 200 bp of intron 1. Sequence analysis indicated that 392 bp of the5′ region (GenBank Accession No. AF029837), shown in FIG. 4, is 77% GCrich and lacks a consensus TATA box sequence. In this Figure, thenucleotides are numbered relative to the ATG translation initiation site(indicated with Met); the coding sequence is indicated in bold.Consensus AP-2, Sp1, and CREB sequences are shown in boxes. Alignment ofthe chick (Matter-Sadzinski et al., EMBO J, 11:4529, 1992) and humanpromoter sequences indicate they share only 52.9% homology. However,consensus Sp1, and AP-2 transcription factor binding sites are presentin both human and chick α7 promoters at approximately the same location,relative to the start of translation (Matter-Sadzinski et al., supra,1992). A CREB consensus binding sequence is present in the humanpromoter, but is not found in the chick.

The primers listed in Table 1 provide a means to obtain sequenceinformation from genomic DNA. Using sequencing techniques standard inthe art (e.g., including, but not limited to standard dideoxysequencing, chain termination sequencing using Taq DNA polymerase orother thermostable polymerases, and automated processes that use theseand other technologies), the sequences near the intron and exonjunctions can be obtained. Such primers have been successfully used toobtain sequence information from blood samples obtained fromschizophrenic patients (i.e., samples obtained as described in Example1). Sequence obtained from this portion of the chromosome also finds usein providing linkage signal for other nicotine-dependent illnessesincluding, but not limited to, small cell lung cancer and juvenilemyoclonic epilepsy. These sequences are then analyzed to determine ifthey contain pathogenic mutations that alter gene function by changingthe amino acid coding, or by altering gene expression or response topromoter molecules, or by introducing variations in gene splicing. Thesemutant sequences are also expressed in transgenic cells in culture or intransgenic mice or in frog oocytes, to determine if they indeed causealtered gene function that produces heritable human illnesses such asschizophrenia.

Example 4 Identification of Expressed Sequence Tagged cDNAs

In this Example, expressed sequence tagged (EST) cDNA clones wereidentified in the EST Database at the National Center for BiotechnologyInformation (NCBI), Bethesda, Md., by BLAST homology searches using α7cDNA specific sequences. Two clones (EST 3952 and EST 52861) werepurchased from Research Genetics and sequenced bi-directionally asdescribed in Example 5. Contigs were constructed using Sequenchersoftware (Gene Codes).

Example 5 Sequence Analyses and Restriction Mapping

In this Example, sequences were determined using standard sequencingkits and automated sequencing. In addition, genomic DNA probed withportions of α7 cDNA was used to order HindIII restriction fragments.

Manual Sequencing

PCR product for hand sequencing was prepared using the ExonucleaseI-Shrimp Alkaline Phosphatase reagent pack (Amersham), per themanufacturer's directions. Sequencing was done using Thermo SequenaseRadiolabeled Terminator Cycle Sequencing Kit from Amersham. Themanufacturer's recommended component concentrations were used with 10 ngof template per 250 bp product per reaction. Reactions were run on aBioRad Sequi-Gene GT sequencing system (BioRad), using a 6%acrylamide/bisacrylamide (19:1) gel.

Automated Sequencing

Plasmids to be sequenced were colony purified, using a Qiagen kit(Qiagen). PCR products from PACS, BACs, and YACs were gel purified usinga Qiagen PCR product gel extraction protocol. Automated sequencing (ABI373 or 377, Perkin Elmer) was conducted using Perkin Elmer ABI DyeTerminator or M13 Dye Primer kits, following manufacturer's protocols.Sequencing was organized into contigs using the Sequencher program (GeneCodes). All sequencing was bi-directional.

Restriction Endonuclease Mapping

Southern analysis of genomic DNA probed with portions of α7 cDNA wasused to order HindIII restriction fragments (Dracopoli et al., supra).DNA was transferred to Hybond N+, and hybridized at 40° C. in 5×Denharts(0.5% SDS, 6×SSC and 50% formamide), then washed twice in 0.1% SDS and0.1×SSC at 65° C. for 10 minutes.

Example 6 Large Insert Clone Contig

Total yeast DNA was isolated from YAC-bearing yeast using a spheroplastmethod (Dracopoli et al., supra). Loci in and around the α7 region werePCR amplified with loci specific primers (i.e., primers shown in Table2, as well as primers for D15S1360 described in Example 2). PCR wasperformed with 1.5 mM MgCl₂: 94° C. for 5 min, 20 cycles of 94° C. for 1min, 56° C. for 2 min, 72° C. for 1 min and 72° C. for 5 min.

Mapping of specific exons was performed using the primers listed inTable 1 and the PCR conditions were 94° C. for 2 minutes, 1 cycle;followed by 35 cycles of 94° C. for 30 seconds, 55° C. for 30 seconds,and then 72° C. for 30 seconds, followed by 72° C. for 7 minutes-1cycle.

Specific amplification was confirmed by sizing the products on agarosegel. PCR products from α7 exons were excised from the gel, Qiagenextracted (Qiagen), and sequenced as described in Example 5.

Additional large insert genomic clones were isolated by PCR screeningwith α7-specific primers (Chumakov et al., supra). YACs 953g6, 948a10,853b12, and 969b11 were isolated from the CEPH YAC Library 3. PAC clones64a1 and 25919 were identified by Genome Systems and BAC 467o18 wasidentified in a BAC library purchased from Research Genetics.

A tentative YAC contig was designed from markers in the YACs andinformation in the CEPH/Genathon Database. YACs providing linkagebetween the full-length and duplicated α7 gene sequences, YACs 895f6,776a12, 791e6, 811b6, 859c11, 801e1, 810f11, 966a4, 764f8, and 822g2,were obtained from Research Genetics. The contig, shown in FIG. 5, wasverified by PCR and sequencing of either α7 sequence or published markersequence. Loci from the 15q13-14 region were assigned to YACs, BACs, andPACs. The results confirmed the presence of markers previously assignedby Genethon (Human Genome Research Center; a publicly accessibledatabase that maintains human genome linkage information). As indicatedin FIG. 5, two allele sizes for the L76630 loci were identified,suggesting that YAC 969B11 spans both α7 nAChr loci.

Exons 5-10 of the α7 nAChR gene and the polymorphic marker L76630 map totwo distinct regions of the contig, suggesting a partial geneduplication. The distal, and full-length, α7 nAChR gene maps close toD15S1360, as indicated by two PAC clones (64a1 and 25919) and one BACclone (467018). Both of these PACs, approximately 120 kb in size,contain the marker D15S1360 which was used to demonstrate linkage ofthis region at 15q14 to a schizophrenic trait. Physical mapping of theα7 gene <120 kb from the linkage marker suggested that the α7 nicotinicreceptor gene is an excellent candidate gene for this trait. Theproximal duplicated exon sequences 5-10 of the α7 nAChR gene map betweenD15S1043 and D15S165. The order of loci was determined to be D15S942,D15S1043, followed by the duplicated sequences L76630, exon 10, exon 9,exon 8, exon 7, exon 6, and exon 5, and then D15S165 and D15S1031. Theclosest marker flanking the 3′ end of the α7 nAChR gene could not beestablished and is either D15S1031 or D15S1010. Thus, the full-lengthgene with the 3′-end closest to D15S1031 has been tentatively oriented,based on the confirmed orientation of the duplicated sequences.

In order to determine if sequence differences were present that mightdistinguish duplicated exons 5-10 from the full-length gene, PCRproducts were generated and sequenced from 11 of the genomic YAC clonesin the contig. Of these 11 clones, two (948a10 and 853b12) clearlymapped to the duplicated region between D15S1043 and D15S165, and eightmapped to the full-length α7 nAChR gene region near D15S1360. All of theα7 exons were found to be present in YACs 776a12, 791e6, 811b6, 953g6,b134h10, 859c11, 810f11 and 801e1. YAC 948a10 contained only exons 5-10,and 853b12, 6-10, while YAC 969b11 appeared to contain both loci. ThisYAC is 1.03 Mb in size (FIG. 5), suggesting that the full-length α7 geneand duplicated sequences are not more than 1 Mb apart.

Sequence variants found in DNA from duplicated and full-length genomicα7 sequences are shown in FIG. 2. In exon 6, a 2 bp deletion wasidentified at bases 497-498 (TG) in clones from the duplicated region,which results in a frame shift in the coding sequence and the insertionof a stop codon within the exon.

Additional sequence variants were found at bases 654, 793, 1269 and 1335of the coding region. These are conservative base changes that do notchange an amino acid. The polymorphic marker, L76630 is also duplicatedas evidenced by the presence of a different number of CG repeats in the3′UT of the full length α7 gene and the 3′ sequences following exon 10in the duplicated sequences. YAC 969b11, which contains both full lengthand duplicated sequences also has two copies of L76630 as does achromosome 15 hybrid, R379-2B2 (See, FIG. 2).

Example 7 RACE Analysis

In this Example, amino terminal clones for the human α7 subunit wereobtained by 5′ RACE (i.e., Rapid Amplification of cDNA Ends; See,Frohman, Amplifications 5:11, 1990), using a kit from Gibco-BRL, withsome modifications. Although some of these products had the aminoterminus nucleic acid sequences that were expected by homology withchicken and rat sequences, some had novel sequences that revealed thepresence of unsuspected alternative exons. The present inventionprovides, for the first time, the sequences of these exons and theirlocation in the genomic structure of α7.

Total RNA was isolated from normal human hippocampus by the method ofChomczynski and Sacchi (Chomczynski and Sacchi, Anal Biochem, 162:156,1987). Briefly, brain tissue from the human hippocampus was disrupted inthe presence of Solution D (4 M guanidium thiocyanate, 25 mM sodiumcitrate pH 7.0, 5% sarcosyl, 0.1 M 2-mercaptoethanol) in a tissuehomogenizer. The homogenized tissue was acidified with 0.1× volume of 2M sodium acetate, pH 4.0, with “X” referring to the initial volume ofSolution D. The acidified tissue homogenate was extracted with 1× volumeof water-saturated phenol and 0.2 volume of chloroform:isoamyl alcohol(49:1). The phases were separated by centrifugation (the supernatantcontains RNA whereas the DNA and proteins remain in the interphase andthe phenol). The RNA was precipitated by addition of an equal volume ofisopropanol (20° C.), followed by centrifugation. The RNA pellet wassubsequently resuspended in 1 mM EDTA, pH 8.0. The concentration of theRNA was determined by measuring the absorbance at 260 and 280 nm.

The first strand cDNA synthesis for 5′-RACE was performed as indicatedin the manufacturer's instructions, with the addition of methylmercurichydroxide (7 mM) to reduce secondary structure. The cDNA was synthesizedusing a human gene-specific antisense oligonucleotide: 5′-AGGACCCAAACTTCAG-3′ (SEQ ID NO:48), complementary to 5′-sequence in the longesthuman clone from the primary cDNA screen. Following cDNA synthesis,terminal deoxynucleotide transferase was used to attach homopolymeric(dCTP) tails to the 3′ ends of the cDNA. A nested gene specificantisense primer and an anchor primer from the 5′-RACE kit, bothcontaining triplet repeat sequences for annealing to the pAMP1 vector,were used for PCR amplification of a homopolymeric, tailed cDNA product.The sequences of the primers were: for the antisense primer,5′-CAUCAUCAUC AUCCAGCGTA CATCGATGTA GCAGGAACTC TTGAATAT-3′ (SEQ IDNO:49), and the anchor primer 5′-CUACUACUAC UAGGCCACGC GTCGACTAGTACGGGIIGGI IGGGIIG-3′ (SEQ ID NO:50). In this anchor primer sequence,the “I” is inosine.

Briefly, the final composition of the PCR reaction for amplification ofdC-tailed cDNA was as follows: 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5mM MgCl₂, 400 nM for both primers, 200 μM each dNTP, 8% DMSO and 0.2unit/μl Taq DNA polymerase. The PCR program was as follows: 94° C., 1min; 57° C., 30 sec; 72° C., 2 min for 35 cycles; final extension at 72°C. for 10 min, then soak at 4° C.

PCR products were Glassmax (Gibco-BRL) purified and reamplified with thesame reaction conditions using the following program: 94° C., 1 min; 50°C., 30 sec; 72° C., 2 min for 5 cycles; 94° C., 1 min; 55° C., 30 sec;72° C., 2 min for 35 cycles; extension at 72° C. for 7 min, and soak at4° C. Products from this PCR reaction were then gel purified and clonedinto the pAMP1 vector (Gibco-BRL) with uracil DNA glycosylase accordingto manufacturer's directions, for subsequent automated sequencing, asdescribed in Example 5.

A group of novel exons located in YAC, PAC and BAC clones containing thefull-length gene and/or the duplicated α7 sequences was also evidenced.These novel exons were discovered in the process of comparing RACEclones, isolated during cloning of the α7 human cDNA, with EST cDNAclones (EST 3952 and EST 52861) found in the EST Database (NCBI) byhomology screening. During cloning of the 5′ end of the α7 codingregion, the RACE technique was used to amplify the 5′ end of the α7 cDNA(Frohman, supra, 1990). Although cDNA clones which matched sequence forpublished human α7 from a neuroblastoma cell line SH-SY5Y (Peng et al.,Mol. Pharm., 45:546, 1994), were obtained, clones with 5′ sequence thatcould not be identified were also obtained.

When EST cDNA clones were subsequently found in the EST database byhomology screening, several were identified that had exons 5-10 andunknown sequence 5′ of exon 5. Comparison of the 5′ ends of the RACE andEST products showed that the novel sequences are partially homologous.PCR primers were designed to these novel sequences for amplificationfrom genomic DNA. Intronic sequence and consensus splice junctions thatidentified these sequences as four alternatively spliced and previouslyunreported exons were then identified. The sizes and splice junctionsfor these novel exons, designated as α7D, α7C, α7B, and α7A are shown inFIG. 6. In this Figure, the sequence of the RACE clone (GenBankAccession No. AF029838) is shown in uppercase, while intron boundariesare shown in lowercase, and are not included in the nucleotidenumbering. The sizes of the exons are indicated below the exondesignations. RACE clones, containing these novel exons were previouslydeposited with GenBank (RACE D-C-B-A-5-6; AF029838; RACE D-C-A-5-6,GenBank Accession No. AF029839).

Example 8 RT-PCR Analysis

Total RNA was isolated from normal human hippocampus, human cingulategyrus, the SH-SY5Y neuroblastoma cell line, and human immortalizedlymphocytes with TRIzol reagent (Gibco-BRL) following manufacturer'sinstructions. The mutations seen in the PAC, BAC, YAC and published α7sequences were screened in seven normal subjects and SH-SY5Y cells. DNAwas evaluated for all subjects, while cDNA was evaluated in exons 1-10and exons 5-10 for all subjects, and exons D-10 were evaluated in onenormal subject and SH-SY5Y cells. The DNA and RNA were obtained asdetailed above. The cDNA was generated as previously detailed.

Total RNA was isolated from normal human hippocampus, human cingulategyrus and SH-SY5Y neuroblastoma cell line by the TRIzol reagent(Gibco-BRL) following manufacturer's directions. RNA was stored as anethanol precipitate until centrifugation and resuspended in 1 mM EDTA,pH 8.0 prior to cDNA preparation.

Total RNA was reverse transcribed at 42° C. for 60 min in a 40 μl volumewith Superscript II reverse transcriptase (Gibco-BRL) and random hexamerprimers (Pharmacia). The final concentration of the components of thereaction were as follows: 1× first strand buffer (50 mM Tris-HCl, pH8.3, 75 mM KCl, 3 mM MgCl₂), 1 mM dATP, 1 mM dGTP, 1 mM dCTP, 1 mM dTTP,8 μM random hexamers, 10 mM DTT, 0.5 U/μl placental Rnase inhibitor(Boehringer-Mannheim), 2.5 U/μl Superscript II reverse transcriptase and500 ng of total RNA.

Primary PCR of the exon 1-10, exon 5-10 and exon D-10 products wasperformed using the Advantage-GC cDNA PCR kit (Clontech). Briefly, 5 μlof RT products were diluted to 50 μl with 40 mM Tricine-KOH, pH 9.2 at25° C., 15 mM KOAc, 3.5 mM Mg(OAc)₂, 5% DMSO, 3.75 μg/ml BSA, 0.2 mM ofeach dNTP, 0.2 μM of each primer, 1 M GC-Melt and 1× Klentaq-1 DNApolymerase mix. Samples were incubated in a Perkin-Elmer 480 DNAThermocycler.

For Exon 1-10, the sense primer was 5′-CGCTGCAGCT CCGGGACTCA ACATG-3′(SEQ ID NO:51), and the antisense primer was 5′-TGCCCATCTG TGAGTTTTCCACATG-3′ (SEQ ID NO:52). The PCR conditions were 94° C., 1 min; 5 cyclesat 94° C., 30 sec, 72° C., 3 min; 5 cycles at 94° C., 30 sec, 70° C., 3min; 25 cycles at 94° C., 20 sec, 68° C., 3 min; final extension at 68°C., 3 min and soak at 4° C.

For Exon 5 to 3′UT α7 transcript, the sense primer was 5′-TGACGCCACATTCCACACTA A-3′ (SEQ ID NO:53), and the antisense primer was5′-CCCCAAATCT CGCCAAGC-3′ (SEQ ID NO:54). The PCR conditions were 5cycles at 96° C., 1 min, 50° C., 30 sec, 72° C., 1 min; 30 cycles at 95°C., 30 sec, 62° C., 20 sec, 72° C., 30 sec; final extension at 68° C., 3min and soak at 4° C.

For Exons D-10, the sense primer was 5′-CTCGGTGCCC CTTGCCATTT-3′ (SEQ IDNO:55), and the antisense primer was 5′-CCTTGCCCAT CTGTGAGTTT TCCAC-3′(SEQ ID NO:56). The PCR conditions were 94° C. 1 min, 5 cycles 94° C.,30 sec, 70° C., 3 min 5 cycles 94° C. 30 sec, 68° C., 3 min, 25 cycles94° C. 20 sec, 66° C. 3 min 1 cycle 68° C. 3 min, cool to 4° C.

The products generated from exons 1-10, 5-10 and D-10 were furtheramplified to incorporate M13 primer sequences into products small enoughto sequence in both directions. PCR conditions were as follows for allsecondary, nested PCR amplifications. Perkin-Elmer Core reagents wereused in standard concentrations using 2 mM MgCl₂, 0.1 mM each dNTP, 1.5U Taq Gold, 10% DMSO and 25 pM of each primer in a 50 μL reaction. PCRreactions were heated at 96° for 5 min, then 5 cycles were performed at96° C. for 1 min, 60° C. for 30 sec, 72° C. for 1 min; then 30 cyclesfor 95° C. for 30 sec, 68° C. for 20 sec, and 72° C. for 30 sec,followed by a 7 min 72° C. extension and cooling at 4° C.

All cDNA reactions were performed in duplicate using 50 ng RNAequivalents in a primary reaction, encompassing the full cDNA length ofinterest, then reamplified in nested, secondary PCR reactions toincorporate M13 primers into shorter products. DNA amplifications wereperformed in duplicate from 100 ng of needle-sheared template, withinexon boundaries. The duplicates were then pooled, purified with aCentricon 100 (Amicon) column, and sequenced using standard M13 DyePrimer chemistry on an ABI 373 Automated sequencer. All templates weresequenced bi-directionally, except where sequence length did not allow anested primer. Alternate splice products were hand called from theelectropherograms. Clean sequences were aligned and checked withSequencher Software (Gene Codes Corporation).

DNA products were generated with primer pairs 1552/1553, 1101/1102,1097/1098 and 1099/1100 to check the 497-498 deletion, 654/690, and1269/1335 mutations, respectively. These primers are shown in Table 3,below. In this Table, “1ry” and “2ry” refer to the first and secondprimer sets in nested PCR. The cDNA amplifications required three setsof primary amplifications, exons 1-10, exons 5-10 and exons D-10. Primerpair 1381/1382 was used to amplify exons 1-10; primers 1482/1483,1101/1098 and 1099/1481 were then used as nested primers from thisprimary PCR to check 497-498, 654/693 and 1269/1335 respectively. Theexon 5-10 product was amplified with primer pair 1502/1503, nestedprimers 1502/1483, 1101/1098 and 1099/1481 were used to check497/498/654/693, 654/690 and 1269/1335 respectively. Exons D-10 wereamplified with primers 1569/1562, and the nested primers 1553/1098 and1097/1481 were used to check 497-498/654/690 and 1269/1335,respectively. Redundancy in the overlap of the secondary PCR productswas used to double check some mutations, necessary when alternatesplicing or base pair deletions occurred, making some base callsdifficult.

Exon 3 codes for 15 amino acids near the amino terminal, in theextracellular domain. An alternate transcript without this exon appearsin most PCR amplifications of this region, at a somewhat diminishedconcentration in comparison to the full-length transcript.

To determine if the exon 5-10 copy of α7 was expressed, a second RT-PCRproduct was generated, encompassing only exons 5-10. The bases whichappeared to be heterozygous in the DNA, but which are not heterozygousin the exon 1-10 transcript, are now fully accounted for in the 5-10exon product, showing the exon 5-10 gene to be expressing as cDNA. Thebase changes fall into three categories, those seen only in thefull-length 1-10 transcript, those changes present only in the 5-10transcript and bases changes seen in both transcripts.

The TG deletion at 497-498 is only present in the 5-10 transcript; the Cat 654 can be assigned to the 1-10 transcript, the T to the 5-10transcript; the G at 933 can be assigned to both transcripts with an Ain some subjects' 1-10 transcript and at 1335 the T can be assigned tothe 1-10 transcript. The base changes seen at 690 and at 1269 appear tobe present in both copies of the gene. These data are consistent withthe base changes seen in the YAC, PAC and BAC clones, and the assignmentof each clone to the duplicated or original gene.

TABLE 3 Primer Sequences Primer # Description Sequence SEQ ID NO: 1097sense/m13fwd+ CCCAGTACTTCGCCAGCACCATGAT SEQ ID NO: 57 1098antisense/m13rev+ CCCCGTCGGGGTCGTGGTGGTGGTA SEQ ID NO: 58 1101sense/m13fwd+ TCCCCGGCAAGAGGAGTGAAAGGTT SEQ ID NO: 59 1102antisense/m13rev+ ACACCAGCAGGGCGAGGGCGGAGAT SEQ ID NO: 60 1099sense/m13fwd+ GACCAGAGTCATCCTTCTGAACTGG SEQ ID NO: 61 1100antisense/m13rev+ TTTCAGGTAGACCTTCATGCAGACA SEQ ID NO: 62 1553sense/m13fwd+ CGATGTACGCTGGTTTCCCTTTGAT SEQ ID NO: 63 1552antisense/m13rev+ TTCCCACTAGGTCCCATTCTCCATT SEQ ID NO: 64 1382 sense/1rycDNA CGCTGCAGCTCCGGGACTCAACATG SEQ ID NO: 65 1381 antisenseTGCCCATCTGTGAGTTTTCCACATG SEQ ID NO: 66 1502 sense/1ry cDNATGACGCCACATTCCACACTAA SEQ ID NO: 67 1503 antisense CCCCAAATCTCGCCAAGCSEQ ID NO: 68 1569 sense/1ry cDNA CTCGGTGCCCCTTGCCATTT SEQ ID NO: 691562 antisense CCTTGCCCATCTGTGAGTTTTCCAC SEQ ID NO: 70 m13sense/extension TGTAAAACGACGGCCAGT SEQ ID NO: 71 m13 antisense/extensionCAGGAAACAGCTATGACC SEQ ID NO: 72 1482 sense/m13fwd+/2ryAAGGAGCTGGTCAAGAACTACAATC SEQ ID NO: 73 1483 antisense/m13rev+CCGGAATCTGCAGGAAGCAGGAACA SEQ ID NO: 74 1101 sense/m13fwd+/2ryTCCCCGGCAAGAGGAGTGAAAGGTT SEQ ID NO: 59 1098 antisense/m13rev+CCCCGTCGGGGTCGTGGTGGTGGTA SEQ ID NO: 58 1502 sense/2ry cDNATGACGCCACATTCCACACTAA SEQ ID NO: 67 1483 antisense/m13rev+CCGGAATCTGCAGGAAGCAGGAACA SEQ ID NO: 74 1553 sense/m13fwd+/2ryCGATGTACGCTGGTTTCCCTTTGAT SEQ ID NO: 63 1098 antisense/m13rev+CCCCGTCGGGGTCGTGGTGGTGGTA SEQ ID NO: 58 1097 sense/m13fwd/2ryCCCAGTACTTCGCCAGCACCATGAT SEQ ID NO: 57 1481 antisense/m13revCCAGGCGTGGTTACGCAAAGTCTTTG SEQ ID NO: 75 1099 sense m13fwd+/2ryGACCAGAGTCATCCTTCTGAACTGG SEQ ID NO: 61 1481 antisense/m13rev+CCAGGCGTGGTTACGCAAAGTCTTTG SEQ ID NO: 75

An RT-PCR product was generated from exon α7D to exon 10 from one normalbrain and from SH-SY5Y cells. The resulting cDNA product containedalternate splice products with exons shown in FIG. 2. The 2 base pairdeletion seen at bases 497-498 in the DNA that is not present in theexon 1-10 transcript was seen in the D-10 transcript, while all of theD-10 product in SH-SY5Y was deleted at 497-498, and subject SL061 washeterozygous for the deletion in the D-10 product. The presence of the Tat base 757 connects this base change to the TG deletion. The G at 690was not expressed in either cDNA. The A at 933 was not present in theminus TG strand of SH-SY5Y. The T at 1296 was expressed in subjectSL061. These products, in subject SL061 cannot differentiate between theexon 5-10 product splicing to exon D versus exon 1, however the productin D-10 from SH-SY5Y can, since only the minus TG strand was expressed,negating the possibility that exons 5-10 from the 1-10 gene are splicingto exon D.

These new exons have been designated as 3′α7A, α7B, α7C, α7D 5′. TheRACE products were variable in their inclusion of Exon B, similar to theEST clones. However, PCR products including exons D-10 gave manyalternate splice products between exons D, C, B, 5 and 6. This samephenomenon was seen in the exon 1-10 transcripts between exons 2 and 6.Based on these results, it was not possible to fully evaluate whetherany of the D-10 transcript contain only exons 5-10 from the duplicatedregion or if this transcript contains some splicing of 5-10 from the1-10 full gene sequence, since the cell line and the brain gavediffering results. Subcloning is used to fully evaluate the base changesto separate the various splice products.

These results indicate that the primer sequences described herein can besuccessfully used to screen both genomic DNA and mRNA for the presencein DNA and the expression in mRNA of sequences which are polymorphic(i.e., different) between individuals. Standard automated and manualsequencing methodologies are used to locate differences in samplesobtained from individuals. It is contemplated that some of thesepolymorphisms, as well as others, have pathogenic roles. Thesepolymorphisms are also used to relate the inheritance of specificalleles of α7 genes through families to the presence of illness orphysiological dysfunction, using standard methods known in the art forlinkage analysis.

Example 9 Single Strand Conformation Polymorphism (SSCP) Analysis

PCR products, <200 bp, containing a single sequence variant wereamplified with ³³Pγ-ATP kinased primer sets using Promega T4 kinase asknown in the art (See e.g., Dracopoli et al., supra). The primers usedin this Example were:

TABLE 4 Primers Used for SSCP Analysis Exon and Primer # Sequence SEQ IDNO: Exon 6b/ #1243 GATGTGCAGCACTGCAAACAA SEQ ID NO: 76 Exon 6b/ #1144TTAAAGCTTGCCCAGGAATAG SEQ ID NO: 77 G Exon 6d/ #1124GGAACTGCTGTGTATTTTCAG SEQ ID NO: 78 C Exon 6d/ #1245AAGACCAGGACCCAAACTTGT SEQ ID NO: 79 Exon 7d/ #1143:GCTTGTGTGTGGTATACACAT SEQ ID NO: 80 TG Exon 7/ #675 GTAGAGTGTCCTGCGGCSEQ ID NO: 81 Exon 10(1438)/ #672 GGTCCGCTACATTGCCAA SEQ ID NO: 82 Exon10/ #593 TGATGGTGAAGACCGAGAAGG SEQ ID NO: 83

Products, denatured with loading dye (7.26 M urea, 60% formamide, 22 mMEDTA, 32 mM NaOH, 0.25% bromophenol blue, 0.25% xylene cynol), wereanalyzed on GeneAmp detection gels (Perkin Elmer) run at both 6° C. and25° C., using Bio Rad PowerPac 3000 with a temperature probe, asdescribed by the manufacturer.

Thus, the frequency of these sequence variants was examined, using SSCPin a group of 43 normal control subjects with no history of mentalillness. Primer sets derived from the exon and intron-exon boundarysequences are used to amplify 200 bp portions of the gene fromindividuals with schizophrenia and their relatives, in order to identifysequence changes that affect gene function. Sequence changes that arenot known to affect gene function, but can serve as markers to traceheritability of particular gene regions through families, are alsoidentified in this process. The −2 bp deletion and the heterozygositiesat 654, 690, 1269, at 1335 were found in this Example.

Almost all subjects were heterozygotic at positions 654 and 690.Nucleotide positions 1269 and 1335 were also found to be polymorphic,suggesting that the duplicated sequences have diverged since theduplication event.

These results indicate that the primer sequences described herein can besuccessfully used to screen genomic DNA in SSCP, a standard genomescreening technique, for polymorphic differences in DNA sequencesbetween individuals. It is contemplated that these polymorphisms, aswell as others, have pathogenic roles. These polymorphisms are also usedto relate the inheritance of specific alleles of α7 genes throughfamilies to the presence of illness or physiological dysfunction, usingstandard methods known in the art for linkage analysis.

Example 10 Electrophysiological Recording, Linkage Analysis, andNonparametric Methods

Electroencephalographic activity was recorded at the vertex andelectrooculographic activity was recorded from the superiororbital-lateral canthus. Five averages of sixteen responses each topaired clicks were obtained, using standard methods (See, Griffith etal., Psychophysiology, 32:460, 1995). The P50 responses weredistinguished from pre-stimulus activity for both normals andschizophrenics at a high level of significance (P<0.001). The averageswere reviewed by two investigators, blind to genetic information, whorejected any average containing excessive electrooculographic activity,drowsiness, startle, or other artifacts; the remainder were combinedinto a grand average, from which the P50 amplitudes were measured andtheir ratio (second response/first response) was calculatedautomatically by a computer algorithm (Nagamoto et al., Biol Psychiat,25:549, 1989). Seven subjects were not used, because artifact-freeaverages could not be selected from their recordings. Recordings wereinitially performed, then repeated approximately three years later. Theearlier recordings were reanalyzed for 2 subjects who were laterdeceased, for 10 subjects who refused repeat recording, and for 2patients who were later on atypical neuroleptics, which can normalizethe P50 ratio; other neuroleptic medication do not affect the phenotype(Nagamoto et al., Biol Psychiat, 40:181, 1996).

Parameters for lod score analyses of P50 ratios were determined from thedistribution of values in 43 unrelated normal individuals and 36unrelated schizophrenic patients (Waldo et al., Schizophr Res, 12:93,1991) and from observations of the segregation of P50 ratios in the ninemultiplex schizophrenic families (i.e., the families described inExample 1). Elevated P50 ratios were defined as values greater than orequal to 0.50, which were found in 91% of the unrelated schizophrenicsand 6% of the normals. Of the remaining unrelated schizophrenics, mosthad values between 0.41 and 0.49, a range therefore coded unknown forthe linkage analysis. If this unknown range was extended to includevalues between 0.40 and 0.60, the results were changed substantially(e.g., lod scores were decreased by an average of 0.54 across themarkers in the 15q13-14 region due to the loss in information). For lodscore analyses, frequency of a gene for abnormal P50 ratio was fixed at0.05, penetrance for the normal genotype was fixed at 0.01, andpenetrance for the abnormal genotypes was fixed at 0.8 (Coon et al.,Biol. Psychiat., 34:277, 1993). These parameters result in a morbidityfor abnormal P50 ratio of 8.7% and a phenocopy rate among abnormalsubjects of 10.4%. The FASTLINK version of the LINKAGE program was usedto compute lod scores at various recombination fractions, Θ (Lathrop etal., Proc Natl Acad Sci USA, 81:3443, 1984). No significantheterogeneity was found using the HOMOG program (Ott, Analysis of HumanGenetic Linkage, Johns Hopkins Univ. Press, Baltimore, 1991). The chanceof false positive lod score results was determined using SLINK (Ott,Proc Natl Acad Sci USA, 86:4175, 1989); 1000 replicates of the pedigreeswere simulated, assuming no linkage to the marker under analysis. Lodscore analysis was performed for each replicate under the dominantmodel; the highest score observed for D15S1360 and P50 under theassumption of no linkage was 1.87.

Sibling pair analysis was performed using the SIBPAL program (Elston,SIBPAL, Statistical Analysis for Genetic Epidemiology, Louisiana StateUniv. Medical Center, New Orleans, La., version 2.2, 1995). Marker datawere used to estimate the proportion of alleles shared through a commonancestor (i.e., identical by descent) for each possible sibling pairingwithin the linkage families. A test was performed to determine if theproportion of alleles shared was >0.50 for abnormal/abnormal pairs. Tocalculate P values, 1000 replicates of the 9 families were simulated foreach marker to determine empirical distributions. Degrees of freedomwere adjusted downward for non-independence when multiple pairings wereused from the same sibship within a family.

A newly developed method, Nonparametric Linkage, uses information fromall genotyped members of a pedigree to assess the extent of allelesshared identical by descent among all affected individuals. Theresulting statistic was normalized, first by subtracting the expectedsharing score under the null hypothesis (no linkage from the observedscore), and then dividing by the score variance under the nullhypothesis. Thus the statistic is asymptotically distributed as astandard normal variable (Z score) under the null hypothesis.Calculations of Nonparametric Linkage statistics were carried out usingthe GENEHUNTER computer programs (Elston, supra). GENEHUNTER also usesan improvement to a previously described algorithm to perform completemultipoint linkage analysis with a large number of highly polymorphicmarkers in pedigrees of moderate size (Kruglyak et al., Am J Hum Genet,58:1347, 1996). Due to computational constraints, the three largestpedigrees were each split into two parts.

Only one marker, D15S1360, yielded a lod score >3.0 (lod scoremaximum=5.3, theta=0.0, P<0.001). DNA markers flanking D15S1360 alsogave positive lod scores. Multipoint analysis showed a maximum lod scoreat D15S1360 of 5.29. Both the sibpair analysis and nonparametric linkageanalysis gave confirming positive results of similar statisticalsignificance. The sibpair analysis showed 0.70 proportion of D15S1360alleles among siblings with abnormal P50 ratios (T=4.07, P<0.0005). Twopoint results from the non-parametric analysis were most significant forD15S1360 (Z=3.95, P<0.0002). A complete multipoint analysis using ninechromosome 15q markers gave a maximum value at D15S1360 (Z=5.04,P<0.000016).

From the above it should be clear that the present invention providesgene sequences encoding mammalian α7 genes and proteins. The presentinvention further provides compositions and methods for targeted therapydirected to α7 abnormalities.

Example 11 Refinement of the Physical Map of the P50-SchizophreniaLinkage Region

This example provides details of further physical mapping of the regionof chromosome 15q13-q14 that is inherited in subjects with the P50deficit and with schizophrenia. The contig depicted in FIG. 11 includesmultiple bacterial artificial chromosomes, and map locations foradditional expressed sequence tags and markers. The region is defined by30 markers and is estimated to be about 4 Mb in length. The full-lengthCHRNA7 gene, implicated in the P50 deficit in schizophrenia, islocalized at this site between unique markers D15 S1013 and D15S1010.Mapping of α7 exons showed that exons 5 to 10 of CHRNA7 had beenduplicated, along with a large cassette of DNA containing several othergenes, and inserted proximal to the full-length gene. The insertionoccurred next to novel exons D-C-B-A, with which the duplicated α7 exonsare expressed as messenger RNA (dupCHRNA7; GenBank Accession No.AF029838). The novel dupCHRNA7 transcript was detected in multipletissues, including human brain and blood leukocytes.

During development of the present invention, exons D-C-B-A were found tobe both duplicated and expressed with downstream sequences that are notof α7 origin (GenBank Accession No. AA861176). These novel exons werealso found to map on chromosome 3 by hybrid clone panel analysis. It iscontemplated that exons D-C-B-A, contained in clone AA861176, wereduplicated at least once on chromosome 15, with one insertion site nearthe dinucleotide repeat (D15S1043), before the partial duplication ofthe CHRNA7 gene. Ultimately, the large cassette containing α7 exons 5 to10, dinucleotide repeat L76630, and expressed sequence tag WI13983 wasduplicated and inserted proximally, interrupting the duplication ofAA861176. Additional analysis of exons D-C-B-A indicated that exon Dactually contains 2 exons and an intervening sequence. The two newlydefined exons are designated as D′ (proximal) and D (distal). The uniqueDNA sequence between the full-length CHRNA7 gene and dupCHRNA7 isapproximately 1 Mb, and contains a large number of mapped expressedsequence tags and markers. The site of the marker D15S1360, isolatedfrom a YAC containing CHRNA7, has been more precisely mapped to intron2, by examining the sequence available from the National Human GenomeResearch Institute (bacterial artificial chromosomes 717i24 and 198g2).The D15S1360 repeat has been used extensively for genotyping of bothschizophrenic patients and controls in the studies disclosed herein.Only two alleles were ever observed in any one individual. Furthermore,the promoter and exons 1 to 4 of the full-length α7 gene were found onlyin bacterial artificial chromosomes and P1 artificial chromosomescontaining D15S1360, all of which map between D15S1031 and D15S1040.Thus, during development of the present invention, the region 5′ of exon4, containing the promoter region of the full-length α7 gene, wasdetermined not to be duplicated.

Example 12 Subject Selection and Sample Collection for CHRNA7 PromoterAnalysis

Subjects were analyzed in a modified case-control study forpolymorphisms in the core promoter of the full length CHRNA7 gene. Atotal of 298 schizophrenic subjects were available for screening (See,Table 5). The sample contained 188 subjects from the NIMH SchizophreniaGenetics Initiative, including DNA samples from 20 families used in asib-pair analysis positive for schizophrenia (Leonard et al., Am J MedGenet, 81:308-312, 1998). These DNA samples were derived fromlymphoblast cultures in the NIMH collection. Three schizophreniclymphoblast cultures were obtained from Israel, while the remaining DNAsamples were isolated from either postmortem brain or lymphoblastscollected in the Denver Schizophrenic Center (Denver, Colo.). Thesamples collected in Denver included 25 specimens from patients withchildhood onset schizophrenia.

TABLE 5 Subjects Used For Screening of the CHRNA7 Promoter* SourceSubjects Number DNA Source P50 Ratio NIMH SZ 188 lymphoblasts 0 DenverSZ 49 lymphoblasts 34 Denver SZ 33 brain 0 Denver COSZ 25 lymphoblasts18 Israel SZ 3 lymphoblasts 0 Total SZ 298 both 52 Denver Control 152lymphoblasts 151 Denver Control 13 brain 0 Total Control 165 both 151*Abbreviations: NIMH, National Institute of Mental Health GeneticsInitative for Schizophrenia; SZ, schizophrenia; and COSZ,childhood-onset schizophrenia.

Postmortem brain was donated by the family of the deceased through theColorado Uniform Anatomical Gift Act (1968) and collected at autopsy.Hospital and autopsy records were reviewed, and family members andphysicians were interviewed to determine age, sex, cause of death, andmental illness status. Brains were weighed, examined for grosspathological features, and divided sagittally. One hemisphere waspreserved in formalin for neuropathological analysis. The otherhemisphere was sliced coronally into 1-cm slices, from which multipleregions were dissected in blocks, frozen in dry-ice snow, and packagedfor storage at −80° C. (Leonard et al., Biol Psychiatry, 33:456-466,1993). DNA was isolated from cortex, by means of standard methods(Sambrook et al., eds., Molecular Cloning: A Laboratory Manual 3^(rd)edition, Cold Spring Harbor, N.Y.: Cold Spring Harbor Press, 2001). Ofthe 165 control DNA samples used in the study, 152 were isolated fromblood collected in the Denver Schizophrenia Center and had no evidenceof current or past psychosis as determined with a Structured ClinicalInterview for Axis I DSM-IV Disorders-Non-Patient Edition (SCID-I/NP),Version 2.0 (First et al., Biometrics Research Department, New YorkState Psychiatric Institute, 1996). In addition, these controls had aFamily History Research Diagnostic Criteria, 3^(rd) edition, interviewthat showed no evidence of family history of psychosis (Endicott et al.,Research Assessment and Training Unit, New York State PsychiatricInstitute, 1978). All local subjects included in this study providedwritten informed consent by means of forms approved by the University ofColorado Health Sciences Center Internal Review Board.

Auditory evoked potentials were recorded on 151 of the living controls,by published methods (Freedman et al., Schizophr Res, 4:233-243, 1991).Briefly, auditory sensory gating is measured by means of the P50 wave ofthe electroencephalogram response to paired auditory stimuli deliveredin the form of clicks. After the second stimulus, delivered 0.5 secondafter the first, the P50 response is decreased in normal individuals. Inmost schizophrenic subjects, the response to the second stimulus is notas greatly diminished as in controls; in some subjects the secondresponse is larger than the conditioning response. Control subjects withno history of mental illness, generally have P50 ratios of the T/Cresponse amplitudes that are less than 0.50. Although some P50 ratios incontrols are higher, before the development of the present invention, itwas not known what causes this variation.

Ethnicities of all subjects were recorded from self-report or familyinterview and represented three major groups. White subjects accountedfor approximately 65% of the samples from schizophrenic patients and 61%of the controls, and African Americans approximately 31% of theschizophrenic sample and 34% of the control subjects. Hispanicsaccounted for 4% of samples from schizophrenic patients and 5% of thecontrols. All schizophrenic subjects in each family were screened forpolymorphisms to detect the possible presence of different variants inrelated individuals.

Example 13 Mutation Screening of the CHRNA7 Promoter

The α7 gene cluster in the 15q13-q14 linkage region was selected as themost likely candidate gene group for mutation studies, based oninclusion of the linkage marker D15S1360 within intron 2 of thefull-length nAChRα7 gene and the neurobiological evidence describedherein that is consistent with diminished α7 expression or function.Because nonsynonymous changes in the coding region were found to be rareand not associated with schizophrenia, the promoter region of the genewas first examined.

Genomic DNA was isolated from individuals as previously described (Gaultet al., Genomics, 52:173-185, 1998), and 231 bases proximal to the α7ATG translation start site were screened. Single-stranded conformationalpolymorphism (SSCP) analysis and sequence analysis were used to identifypolymorphisms in this core promoter region (Gault et al., supra, 1998).Briefly, two primers sets for overlapping fragments covered the regionfrom bases −14 to −268 (primer sets 4 and 5 of Table 6). The primer setswere phosphorylated with [γ-³³P]-adenosine triphosphate and T4 kinase(Promega), then used separately to amplify the promoter region by PCR.PCR was done with Taq Gold and a GeneAmp PCR System 9600 kit(Perkin-Elmer) using the following program: 95° C., 3 min; 95° C., 30sec; 60° C., 30 sec; 72° C., 30 sec; for 35 cycles; then 72° C., 3 min.The products were denatured with loading dye (7.26M urea, 60% formamide,22 mM EDTA, 32 mM sodium hydroxide, 0.25% bromophenol blue, and 0.25%xylene cyanol) and were separated on GeneAmp detection gels(Perkin-Elmer) run at 4° C. and 25° C. by means of a Bio-Rad Power Pac3000 Power Supply with a temperature probe. The results were similar atboth temperatures. SSCP analysis of DNA samples, from both schizophrenicand control subjects, was completed in the same experiment.

TABLE 6 Primer Sets Used For PCR, DNA Sequencing and SSCP Analysis SizeSEQ ID Sets Sequence (bp) Base Use NO 1 S 5′-GGTTGGCAAGACTTCCGAAGCC-3′618 −553 to −531 PCR, SEQ 126 1 AS 5′-GTGGCTTTACCGTGCAGGAGCG-3′ +44 to+65 PCR, SEQ 127 2 S 5′-AGTACCTCCCGCTCACACCTCG-3′ 271 −269 to −248 PCR,SEQ 128 2 AS 5′-ATGTTGAGTCCCGGAGCTGCAG-3′ −20 to +2 PCR, SEQ 129 3 S5′-CTGGCCAGAGGCGCGAGGCCG-3′ N/A −347 to −327 SEQ 130 4 S5′-GGGGCTCGTCACGTGGAGAGGC-3′ 180 −170 to −149 SSCP 131 4 AS5′-AGCAGCGCATGTTGAGTCCCGGAGC-3′ −14 to +10 SSCP 132 5 S5′-GTACCTCCCGCTCACACCTC-3′ 176 −268 to −249 SSCP 133 5 AS5′-CGGCTCGCGCGCCTTTAAGGA-3′ −112 to −92 SSCP 134 6 S5′-AGTACCTCCCGCTCACACCTCG-3′ 696 −269 to −248 PCR, SEQ 135 6 AS5′-GGAGGCTCAGGGAGAAGTAG-3′ +407 to +427 PCR, SEQ 136 *Abbreviations: S,sense; AS, antisense; and bp, base pairs.

Automated DNA sequencing on an Applied Biosystems 377 DNA Sequencer wasused for verification of polymorphisms and determination of the specificbases changes, as previously described (Gault et al., supra, 1998).Generally, a large fragment of 618 bp was generated with the use ofprimer set 1. A final concentration of 1.25M betaine (Sigma-Aldrich),added to Master Mix 2 in the Expand Long Template PCR System kit (Roche)was used to amplify the fragment, with the following PCR program: 93°C., 2 min; 38 cycles at 93° C., 30 sec; 62° C., 30 sec; 72° C., 1 min;followed by 72° C., 7 min. Briefly, 200 ng of genomic DNA was diluted ina volume of 25 μl to the following final concentrations: 1× Expand LongTemplate PCR Buffer 3 (Roche), containing 0.75 mM magnesium chloride,1.67 U of Expand Long Template enzyme mixture (Taq and Pwo thermostableDNA polymerases), 0.25 mM of each deoxynucleotide triphosphate, 0.4 μMof each primer, and 1.25M betaine. An additional primer set 2 was oftenused for sequencing of a shorter fragment in the proximal promoterregion (271 bp). The PCR conditions for the shorter fragment were thesame as for the longer fragment.

The G/C(−194) and G/A(−191) variants had indistinguishable SSCPpatterns. Samples with these polymorphisms were analyzed with WAVEtechnology (Transgenomics). WAVE detects sequence changes in PCRproducts based on differential separation through temperature-modulatedliquid chromatography and a high-resolution matrix with detection byabsorbance at 254 nm. During development of the present invention, thePCR products generated with primer set 2 were used. An aliquot of thePCR fragment generated from control or patient DNA was then used forheteroduplex formation in the thermal cycler as follows: 95° C. for 5min, ramp slowly from 95° C. down to 25° C. for 45 min, then hold at 4°C. The melting profile of a normal 271 bp promoter sequence wasdetermined with the Wavemaker Program (Transgenomics). A temperaturecurve was generated for the heterozygous samples containing eitherG/C(−194) or G/A(−191) at temperature ranging from 69° C. to 73° C. Theresulting chromatograms showed the presence of heteroduplex peaks thatwere resolved optimally at 71° C. A triethylammonium acetate andacetonitrile gradient specified by the manufacturer was used forelution. All subsequent samples were run under identical conditions.

Approximately 2.6 kb (SEQ ID NO:122) of DNA sequence 5′ of exon 1 in thefull-length CHRNA7 gene was cloned from P1 artificial chromosome 24919that contains CHRNA7 exons 1 to 3 (Incyte). Subclones of this regionwere constructed for determination of functional domains for genetranscription (See, FIG. 12, panel A). Base pair numbering begins with−1 at the position preceding the translation start in exon 1. The threefragments indicated were cloned into the pGL3 Basic Vector (Promega) foranalysis of promoter sequence effects on the reporter gene luciferase. Afragment of 231 bp, immediately 5′ of exon 1, was identified as the corepromoter sequence and is sufficient to drive high levels oftranscription in vitro. Sequences further upstream, included infragments of 1.0 kb and 2.6 kb, were identified as containing putativerepressor elements.

The 231-bp core promoter region is homologous to the bovine α7 corepromoter region, including conservation of some transcription factorconsensus sequences (Carrasco-Serrano et al., J Biol Chem,273:20021-20028, 1998). Thus, the human α7 promoter region iscontemplated to be regulated in part by SpI and AP-4 transcriptionfactors, for which there are 2 clusters of consensus sites (See, FIG. 2,panel B). The regions including the Sp1 sites were also identified asG/C boxes, which are contemplated to bind other transcription factors.There is a consensus serum responsive element (SRE), also found in thebovine gene, but not in chick gene (Couturier et al., Neuron, 5:847-856,1990).

Mutation screening was completed for the 231-bp core promoter in 195schizophrenic individuals and 165 controls, demonstrating a complexcluster of variants (See, Table 7). There were 12 different singlenucleotide changes, including two insertions and a deletion. Many of thevariants lie in putative transcription factor consensus bindingsequences (See, FIG. 2, panel B). For instance, the G/C variant at −194introduces a new SpI site. In addition, some subjects were found tocarry double variants that were combinations of the single variants (8different combinations). The total numbers of single and double variantsfound in control and schizophrenic subjects are shown in Table 7 andTable 8, respectively, stratified by ethnicity. One polymorphism, aninsertion of +CGGG at −140 bp, was found in a single subject with adiagnosis of psychosis, not otherwise specified (DSM-IV, 298.9). As thisdiagnosis was not included in either the control or schizophrenic samplediagnoses, this individual was not included in Table 7 or in thestatistical analysis, but is disclosed to indicate that additional andperhaps more complex polymorphic patterns may remain to be discoveredwith the methods and compositions disclosed herein. Forty-seven of 165control individuals and 71 of 195 schizophrenic patients had one of thesingle polymorphisms. Although one single variant (−93 bp) and twodouble variants (−93 bp/−194 bp and −191 bp/−194 bp) were found only incontrol subjects, a larger number of both single and double variantswere found in schizophrenic patients than in controls. The differencewas not, however, statistically significant. Eight of the 12 variants(−86 bp, −92 bp, −143 bp, −178 bp, 480 bp, −191 bp, −194 bp, and −241bp), marked with asterisks in Table 7, were found to be more prevalentin schizophrenic subjects. Twenty-seven of 165 control subjects had oneof these 8 variants, but 59 were found in the 195 schizophrenicpatients. Association of the single variant −86 bp C/T withschizophrenia in the combined ethnic groups reached significance(P=0.04). This polymorphism was examined alone because −86 bp C/T wasfound to be the most common variant in the region, and because it wasfound to have the highest prevalence in schizophrenic patients. It isfound more frequently in whites than in African Americans. The genotyperelative risk for this variant was 2.39 (95% confidence interval,1.07-5.32). The principal polymorphisms found in African Americanschizophrenic patients were the G deletion at −178 bp and the G/Asubstitution at −191 bp. Although more variants at these sites werefound in schizophrenic subjects than in controls, fewer subjects werecarrying each of the polymorphisms and the differences were notsignificant.

Thirty-four affected full sib pairs were examined in 30 families thathad one or more of the promoter polymorphisms. Fourteen of the 34, or0.41 sib pairs, shared at least one of these variants. For the common−86 bp C/T variant, 6 of 12 sib pairs shared the polymorphism.

TABLE 7 Single Promoter Variants in Control and Schizophrenic SubjectsControl Subjects Schizophrenic Subjects Variant White African HispanicTotal White African Hispanic Total  −46 G/T 0 11 0 11 0 9 0 9   −86 C/T9 0 0 9 20 1 2 23*†  −92 G/A 1 0 0 1 1 1 0 2*  −93 C/G 1 0 0 1 0 0 0 0 −143 G/A 0 0 0 0 0 1 0 1* −172 1 0 0 1 0 0 0 0  +CGGGGG −178 −G 0 3 0 30 9 0 9* −180 G/C 0 0 0 0 0 0 1 1* −190 +G 0 5 2 7 1 2 0 3  −191 G/A 0 30 3 1 6 0 7* −194 G/C 9 2 0 11 12 1 1 14*  −241 A/G 0 0 0 0 2 0 0 2*Total 21 24 12 47 37 30 4 71  Variants Total 103 54 8 165 129 56 10195   Subjects *Found more frequently in schizophrenic subjects. †P =0.04.

TABLE 8 Double Variants in Control and Schizophrenic Subjects ControlSubjects Schizophrenic Subjects Combination White African Hispanic TotalWhite African Hispanic Total −46/−178 0 0 0 0 0 3 0 3* −46/−190 0 0 0 00 1 0 1* −46/−191 0 0 0 0 0 1 0 1* −86/−194 2 0 0 2 1 0 0 1  −86/−241 00 0 0 1 0 0 1* −93/−194 1 0 0 1 0 0 0 0  −178/−191  0 0 0 0 0 1 0 1*−191/−194  0 1 0 1 0 0 0 0  Total 3 1 0 4 2 6 0 8† Variant Total 103 548 165 129 56 10 195   Subjects *Found only in schizophrenic individuals.†P = 0.38 (not significant).

In other embodiments, mutation screening of the 2.6 kb upstreamregulatory region is done with a Transgenomics WAVE™ denaturing highperformance liquid chromatography system (DFPLC). This system detectspattern differences in PCR fragments bearing mutations. Primers aredesigned for overlapping fragments of approximately 300-500 bp from−2600 bp to the proximal promoter. The size of the fragment ranges from100 bp to 300 bp and depends upon the melting profile for the sequence,as determined by utilization of the Transgenomics software. At least 10fragments are screened. The fragments generated from each subject arethen run on the DFPLC system. Fragments showing a pattern different fromthe wild-type are sequenced for identification of the specific mutation.Patterns similar to wild type are mixed with a wild type sample toensure that homozygotic mutations are not missed. These have been rarein the proximal promoter region, but they do exist and this mixingprotocol is used successfully for their detection. As an example of thepattern complexity, representative DFPLC patterns are shown for theproximal promoter mutations in FIG. 17.

Example 14 Analysis of Double Variants

Some subjects were found to have more than one polymorphism in theCHRNA7 core promoter. To determine whether these were on the samechromosome, the two alleles were examined individually by cloning. ThePCR products were generated with the GC-RICH PCR system (Roche), withfinal concentrations of 1× buffer, 2.0 mM magnesium chloride, 0.25 mMdeoxynucleotide triphosphates, and 0.5 μl of enzyme mix in a 25 μlvolume. Three primer sets were used (See, Table 6): primer set 1, corepromoter to intron 1; primer set 2, core promoter only; and primer set6, core promoter to intron 2. The reaction for the smaller product,generated with primer set 2 (0.8 μM concentration of each primer), alsoincluded 1.0M GC-RICH resolution solution, while for the larger productsgenerated with primer set 1 (0.4 μM concentration of each primer) orprimer set 6 (0.4 μM concentration of each primer), 0.8M GC-RICHresolution solution was included. All PCR products were amplified in aPerkin-Elmer 480 PCR thermocycler by means of the following program: 96°C., 3 min; 33 cycles at 96° C., 30 sec; 56° C., 30 sec; 72° C., 7minutes. The appropriate PCR bands were gel-purified with the cONCERTRapid Gel Extraction System (Life Technologies), and cloned into the PCR4-TOPO vector with the TOPO TA Cloning Kit (Invitrogen). Plasmid DNA wasisolated with the S.N.A.P. Miniprep kit (Invitrogen) and analyzed by DNAsequencing. Approximately 20 clones were sequenced for each doublevariant cloned.

In the individuals included in this study, 8 doubly polymorphic patternswere found (subjects had more than 1 polymorphism in the core α7promoter. Five of these double variants were found only in schizophrenicpatients (marked with asterisks in Table 8). DNA fragments were clonedand sequenced from individuals with most of the double variant patternsisolated thus far. Three primer sets of Table 6 were used: 1 thatamplified the core promoter of 271 bp (primer set 2); another set thatamplified the core promoter, exon 1, and part of intron 1 (primer set1); and a primer set that amplified the core promoter, exon 1, intron 1,exon 2, and part of intron 2 (primer set 6). Two variants were neverfound on the same chromosome, and only 2 alleles were present in allcases examined, indicating that the core promoter region is notduplicated in these individuals and, further, that each variant is aseparate allele. Thus, polymorphisms in the core promoter of thefull-length α7 nicotinic receptor gene are found more frequently inschizophrenic individuals than in subjects with no family history ofschizophrenia, and double variants are likely to result from inheritanceof one mutant allele from each parent.

Example 15 Analysis of Promoter Function

Promoter function was determined by means of a luciferase reporter geneassay. To identify a core promoter sequence in the 5′ sequence upstreamof the ATG translation start site in the α7 nicotinic receptor gene,fragments of this region were subcloned into the pGL3-Basic Vector(Promega), using PCR and the pGEM-T Easy Vector System II kit (Promega).Initially, a 2602 bp fragment was inserted into the pGL3 vector (See,FIG. 12 panel A, −2600 to +2). A 1064 bp clone was generated by partialPstI digestion of the original fragment and cloned into the pGL3-BasicVector. PstI was then used to subclone a fragment of 231 bp, containingthe proximal promoter region, which is conserved in the bovine α7 gene(Carrasco-Serrano et al., J Biol Chem, 273:20021-20028, 1998).Transcription factor consensus sequences in the 5′ upstream region wereidentified with the TRANSFAC program available on the internet, courtesyof the Research Group Bioinformatics/AG Bioinformatik. Variantsdiscovered in the mutation screen were introduced into the normal PstIcore promoter clone by using the QuikChange Site-Directed MutagenesisKit (Stratagene). Transient transfections were done with ProFectionMammalian Transfection Calcium Phosphate System (Promega), with thehuman neuroblastoma cell line SHSY-5Y (Flora et al., Eur J Pharmacol,393:85-95, 2000). The SHSY-FY cell line was grown in 1:1 Ham F12:DMEM,and 10% fetal calf serum, plated at 2×10⁵ cells/35 mm plate. Five μgplasmid DNA prepared with EndoFree Plasmid Kits (Qiagen) wascotransfected with 1 μg of pRL-TK Vector (Promega). Cells were harvestedafter 48 hours and luciferase activity was measured with theDual-Luciferase Reporter Assay System (Promega) and a Turner DesignsLuminometer Model TD 20/20.

In vitro functional analysis was performed for several of thepolymorphisms found more frequently in schizophrenic subjects. Aluciferase reporter gene assay was used to compare the normal corepromoter sequence with a fragment containing one of these variants. Asshown in FIG. 13, variants at −86 bp, −92 bp, −143 bp, −178 bp, −194 bp,and −241 bp decreased transcription of the luciferase reporter gene inthis in vitro assay, indicating that presence of one of thesepolymorphisms in the core promoter region decreases transcription fromthe gene. The −86 bp C/T variant resulted in a decrease in luciferasetranscription of 20% (P<0.0001). The functional promoter mutationsexamined thus far were statistically more prevalent (chi-squared₁=7.302,P=0.007) in schizophrenic patients than in the control subjects.

Example 16 Statistical Analysis

For the statistical analysis, total counts from schizophrenicindividuals included polymorphisms detected in only one schizophrenicindividual per family, unless a second mutation was also present inanother affected individual. In this case, the second variant was alsocounted. Subjects homozygous for the common allele were also counted.This strategy was chosen to report the full range of polymorphisms inschizophrenic patients without biasing the results by including multipleindividuals who have the same polymorphism based on common ancestry. ttests were used to compare means. A Satterthwaite t test was used forcomparison of means with difference variances; chi-squared tests andlogistic regression were used to compare prevalence rates. For thedouble variants in the promoter region, cloning experiments indicatedthat each polymorphism is a separate allele.

Although promoter variants were found in control subjects, they werefewer in number than in schizophrenic patients. In complex disorderswhere multiple gene variants may be interacting with environmentalfactors to produce the disease, it has been suggested that functionalpolymorphisms are likely to be common in the general population, whereeach may have a more elementary phenotype, such as a biochemical orelectrophysiologic abnormality that is part of the pathophysiology ofthe illness (Lander and Schork, Science, 265:2037-2048, 2000; andGershon, Biol Psychiatry, 45:551-558, 1999). The association of CHRNA7promoter polymorphisms was examined, in the living control subjects,with a functional electrophysiologic assay (e.g., inhibition of the P50response to paired auditory stimuli). The P50 auditory sensory gatingwas measured in 151 of the 152 live control subjects examined in thisstudy. The range of P50 ratios (T/C) for controls was 0.00 to 1.91.Overall mean P50 ratio was 0.22±0.27. There were 38 adult schizophrenicsubjects examined locally where P50 recording was done. The mean P50ratio for these patients was 0.92±1.02, with a range of 0.00 to 4.96.Eighteen patients with childhood-onset schizophrenia, included in themutation screen, also had their P50 recorded. Their mean was 1.05±0.91,similar to that of the adult patients with schizophrenia. The mean ratiofor the schizophrenic patients was significantly greater than that ofthe control subjects (t₂₀₅=8.49, P<0.0001).

Tracings for subjects with and without the −86 bp C/T polymorphism areshown in FIG. 14. A control subject with the normal C/C genotype had aT/C ratio (P50 ratio) of 0.14, indicating that the test response to thesecond auditory stimulus was being inhibited. However, a control subjectcarrying a −86 bp C/T heterozygotic genotype had a T/C ratio of 0.60,demonstrating a much lower level of inhibition. A schizophrenic patientwith the −86 bp C/T genotype also had a higher T/C ratio of 0.54. Theseresults indicate that the presence of a promoter variant is associatedwith decreased inhibition in the sensory gating paradigm and, hence, ahigher T/C(P50) ratio.

The relationship between the means for the P50 T/C ratios and thepresence of CHRNA7 promoter variants was then examined in the 151control subjects. As shown in FIG. 15 panel A, the mean P50 ratio forcontrols with no CHRNA7 promoter variant was 0.179±0.014. However, themean for control subjects with one of the single or double variants was0.458±0.055. The results were analyzed, using a Satterthwaite t test forsamples with different variances. The control subjects with nopolymorphisms had a significantly lower mean P50 ratio than controlsubjects in whom a promoter variant was found (P<0.0001), demonstratinga strong relationship between the presence of a promoter variant anddecreased sensory processing.

In the patients with adult-onset disease, where P50 had been recorded, 7of 8 polymorphisms in the core promoter were found in schizophrenicpatients with P50 ratios greater than 0.50. In the 18 patients withchildhood-onset disease, wherein P50 had been recorded there were 7polymorphisms, 5 of which were found in subjects with P50 ratios greaterthan 0.50. These results indicate that a similar relationship betweenthe CHRNA7 promoter polymorphisms and the recorded P50 ratio exist inboth adult-onset and childhood-onset schizophrenia. Logistic regressionanalysis of the control data indicated, that the presence of promotervariants is better described by three groups, than by a regression lineon the P50 range (See, FIG. 15, panel B). One group with individualaverage P50 ratios less than 0.20 was found to have stable auditorygating. A second group with P50 ratios between 0.20 and 0.50 was foundto have a less stable filtering mechanism. A third group with P50 ratiosconsistently greater than 0.50 was found to exhibit very little auditorygating, similar to what has been described herein in the schizophrenicpopulation. Control subjects with no polymorphism in the core α7promoter were found to have P50 ratios in the first 2 groups, with mostin the less than 0.20 group. In contrast, controls with polymorphismswere more evenly distributed among the three P50 groups, while onlysubjects with a promoter variant were found to have P50 ratios greaterthan 0.50.

Example 17 Subject Selection and Sample Collection for CHRNA7 anddupCHRNA7 Analysis

Samples from 171 families with schizophrenic members and 185 samplesfrom controls were available for screening. The sample populationincluded 86 families from the NIMH Schizophrenia Genetics Initiative.Sixteen of these families had been used in a sib pair analysis showinggreater than 50% inheritance-by-descent to a dinucleotide markerD15S1360 in the CHRNA7 gene (0.58; P<0.0024) as described (Leonard etal., Am J Med Genet, 81:308-312, 1998). Nine probands from the P50linkage analysis (Freedman et al., Proc Natl Acad Sci USA, 94:587-592,1997) were also included and the remaining samples were collected in theDenver Schizophrenia Center. When postmortem brain samples were used,diagnosis was based upon review of medical records and family andphysician interviews. Of the controls, 166 were interviewed and found tohave no evidence for current or past psychosis, using two differentinterviews (See, First et al., Structured Clinical Interview for DSM-IVAxis I Disorders-Non-Patient Edition, SCID-I/NP, version 2.0, NY:Biometrics Research Department, New York State Psychiatric Institute,1996; and Endicott et al., Family History—Research Diagnostic Criteriainterview, FH-RDC, 3rd edition, NY: Research Assessment and TrainingUnit, New York State Psychiatric Institute, 1978). In addition, auditoryevoked potentials were recorded on controls, using published methods(Freedman et al., Schiz Res, 4:233-243, 1991).

The 84 samples used for cDNA mapping of the eight common variants werecollected in the Denver Schizophrenic Center, and were primarilyobtained from Caucasian subjects. As shown in Table 15, 28 samples fromCaucasian individuals with schizophrenia (18 male and 10 female) and 49samples from Caucasian controls (35 male and 14 female) were analyzed.

TABLE 15 Subjects Used For Screening the CHRNA7 and dupCHRNA7 GenesSubjects Schizophrenics Controls Ethnicity tissue Total white BlackTotal white Black brain 28 25 3 20 19 1 lymphocytes 4 3 1 32 30 2 totals32 28 4 52 49 3

TABLE 16 PCR Primers for Amplification of the CHRNA 7 Gene SEQ IDProduct Variants NO Strand Primers T_(A) SSCP Exon 1 45, +82 137 SGCGGCGAGGTGCCTCTGT 60° C. 25° C. 138 AS GGATCCCACGGAGGAGTGGAG Exon 2 139S CCTGCCCGGGTCTTCTCTCCT 58° C. 25° C. 140 AS AACTAGAGTGCCCCAGCCGAGCTExon 3 141 S AACAACGCTCTCGACAGTCAGATC 58° C. 25° C. 142 ASAAGATCTTGCAGCCCATGGGAG Exon 4 334 143 S GGAATTCTCTTTGGTTTTGCAC 58° C.6° C. 144 AS ACATATCCAGCATCTCTGTGA Exon 5 370 145 STCATGCAGTCCTTTTCCTGTTTC 60° C. 6° C. 146 AS CTCGCTTCAGTTTTCTAACATGG Exon6 147 S GGAACTGCTGTGTATTTTCAGC 58° C. both 148 AS TTAAAGCTTGCCCAGGAATAGGExon 7 149 S GCTTGTGTGTGGTATACACATTG 58° C. both 150 ASTCCAGAGCTGATCTCAGCAGAAG Exon 8 861 151 S GAGGAACGGCTGTGTGTTTAT 58° C.25° C. 152 AS CTGGGCACACTCTAACCCTAACC Exon 9 153 STGTGACGTGCAGTGCCACAGGA 60° C. 25° C. 154 AS AAACCCTAGGAGGAGCCTCCTT Exon10 155 S GATCAGCCCGTTTCCGCCTCAG 58° C. both 156 ASCCGATGTACAGCAGGTTGCCGTTGC Exon 6* 497-8 157 S CAGTACCTGCCTCCAGG 58° C.25° C. 158 AS TCCAAGGACGAGCCTCCGTAAGA Exon 7* 654/690 159 SCTATGAGTGCTGCAAAGA 58° C. 25° C. 160 AS CAGGGGATCAGCAGGTT Exon 7*698/+21 161 S GCCGCAGGACACTCTAC 58° C. 25° C. 162 ASTCCAGAGCTGATCTCAGCAGAAG Intron 7* −11, −20, −29 163 SGCCCCTCGTTAGACAGAATTGAG 58° C. 25° C. 164 AS CTGGGCACACTCTAACCCTAACCExon 10* 1044, 1116 165 S GATCAGCCCGTTTCCGCCTCAG 58° C. 25° C. 166 ASCCGATGTACAGCAGGTTCCCGTTGC Exon 10* 1335 167 S TCCCGACCCCCGACTCT 58° C.6° C. 168 AS TGATGGTGAAGACCGAGAAGG Exon 10* 1269, 1354, 169 STCCCGACCCCCGACTCT 58° C. 25° C. 1456 170 AS TGATGGTGAAGACCGAGAAGG Exon10* 1466 171 S CCTTCTGGGTCTTCACCATC 58° C. 25° C. 172 ASGCCTCCACGAAGTTGGGAGC Exon 10* 1487 173 S GGTCCGCTACATTGCCAA 58° C.25° C. 174 AS CCTTGCCCATCTGTGAGTT 3′UT* 1737, 1837 175 SGTGTTGCTTACGGTTTCTT 58° C. 25° C. 176 AS TTTCAGGTAGACCTTCATGCAGACA cDNA#177 S TGCCCATGTGTGAGTTTTCCACATG 72-68° C. 1-10 178 ASCGCTGCAGCTCCGGGACTCAACATG cDNA# 179 S CTCGGTGCCCCTTGCCATTT 72-68° C.D-10 180 AS CCTTGCCCATCTGTGAGTTTTGCAC

Example 18 Mutation Screening of the CHRNA7 and dupCHRNA7 Genes

All schizophrenic subjects in each family were screened forpolymorphisms to detect the presence of different variants in relatedindividuals. Initially, a strategy was used to screen genomic DNA from96 samples from individuals where postmortem brain tissue orlymphoblasts were available. This was done because mRNA would be neededfor the mapping of variants to either the full-length CHRNA7 or itsduplication (dupCHRNA7). In the initial gene mutation screen, all theexons, intron/exon boundaries, and the 3′untranslated region (UT) wereexamined by means of single-strand conformation polymorphism (SSCP)analysis using the primers shown for exons 1-10 in Table 16. Exon 10 andthe 3′ UTR were divided into an additional eight overlapping PCRfragments of approximately 200 bp, designed from the CHRNA7 sequence(GenBank Accession No. U40583). For SSCP analysis the primer sets werekinased using [γ-³³P] ATP with Promega T4 kinase, then used to amplifyregions of the CHRNA7 gene by PCR. Briefly, PCR was done using Taq Gold™and GeneAmp® PCR System 9600 (Perkin-Elmer, Foster City, Calif.) withthe following program: 95° C. for 3 min; then 35 cycles of 95° C. for 30sec, 58° C. for 30 sec, and 72° C. for 30 se; followed by 72° C. for 3min. Specific annealing temperatures (T_(A)) are provided in Table 16).The products, amplified and analyzed separately, were denatured withloading dye (7.26 M urea, 60% formamide, 22 mM EDTA, 32 mM NaOH, 0.25%bromophenol blue, 0.25% xylene cyanol), and separated on GeneAmpdetection gels (Perkin-Elmer) run at 25° C. and 6° C. using a BioRadPower Pac 3000 with a temperature probe. Samples with unique SSCPpatterns were sequenced and polymorphisms were correlated with the SSCPpatterns. Identified variants were subsequently screened in additionalgenomic samples from controls, individuals with schizophrenia, andfamily members, using the appropriate primers and gel conditions. InTable 16, additional primer sets used to detect specific variants areindicated with an asterisk, while primers used in primary RT-PCR formapping are indicated with a pound sign.

Mutation analysis of the α7 nicotinic receptor gene CHRNA7 and itspartial duplication dupCHRNA7 was carried out using SSCP, and sequenceanalyses. FIG. 16 panel A depicts the 15q13-q14 region containing CHRNA7and dupCHRNA7. The unique dinucleotide marker D15S1360, used in severallinkage studies (Freedman et al., Am J Med Gen, 105:794-800, 2001;Freedman et al., Proc Natl Acad Sci USA, 94:587-592, 1997; Leonard etal., Am J Med Genet, 81:308-312, 1998; and Freedman et al., Am J MedGen, 205:20-22, 2001), lies in intron two of CHRNA7 (Leonard et al.,Arch Gen Psychiatry, 59:1085-1096, 2002). D15S1031 and D15S144, (alsosingle copy) flank the full-length CHRNA7 gene and duplicated cassette(duplicon). Unique loci D15S1043 and D15S165 flank the proximalduplicon. The duplicon contains exons 5-10 of the CHRNA7 gene, thedinucleotide repeat L76630, exons D′-D-C-B-A, and the Expressed SequenceTag (EST) WI13983. The transcripts from both α7 containing genes areshown in FIG. 16 panel B, with their unique 5′ ends and the number ofvariants mapped to each exon. The orientation of the duplicon is shownas head to tail, determined from yeast artificial chromosome (YAC)mapping from two separate YAC libraries (Gault et al., Genomics,79:197-209, 2002). A head to head orientation has been reported based onBAC clone mapping from a single library (Riley et al., Genomics,79:197-209, 2002), suggesting that the orientation of this duplicon maybe polymorphic.

Thirty-three variants in the CHRNA7 gene cluster were identified ingenomic DNA from individuals with schizophrenia and controls ofCaucasian, African American and Hispanic descent (Tables 17, 18 and 19).Twenty-one different variants were found in the coding region of the α7genes, including 10 non-synonymous variants. Base pair numbering is fromthe first base pair in exon 1. Allele frequencies for 14 of the rarevariants were calculated and are shown in Table 20. Allele frequenciesfor the more common variants were not determined because they could behomozygous in either dupCHRNA7 or full-length CHRNA7 genes. Six variantswere found more frequently in the African Americans than the Caucasians(Table 21). Three variants at: 497-8 bp (2 bp deletion), 654 bp, and1466 bp, were found more frequently in Caucasians than in AfricanAmericans. Two rare, but non-synonymous variants in Exon 5 at 370 bp,and in Exon 7 at 698 bp, were found only in Hispanics (See, Table 17).

In Tables 17-21, the following nomenclature and abbreviations apply: E,exon; I, intron; V, number of individuals with the variant; T, totalnumber of individuals; α7, full length gene; and dα7, duplicate gene.Numbering for exons and 3′UT is from the ATG start, while numbering forintrons is from the 5′ donor splice site (+) or 3′ acceptor splice site(−). Variants: ^(a)exon 4, I112V; ^(b)exon 5, A124T; ^(c)exon 6, 2 bpdeletion at L166>in Caucasian subjects X²=48.66, 1, P<0.0001; ^(d)exon7, Y233C; ^(e)exon 9, G324R; ^(f)exon 10, S372R; ^(g)exon 10, E452K;^(h)exon 10, 1486V; ^(i)exon 10, S489L; ^(j)exon 10, A496D; ^(k)intron9, X²=9.986, 1, P=0.0016; and ¹provisional mapping.

TABLE 17 Non-synonomous Variants Identified in CHRNA7 and dupCHRNA7Schizophrenics Controls Afr. Afr. Cauc. Am. Hisp. Cauc. Am. Hisp. MapSite bp change V T V T V T V T V T V T α7 dα7 E4^(a)  334 A→G 0 113 1 430 6 0 103  0 55 1 8 X E5^(b)  370 G→A 0 112 0 42 1 7 0 100  0 53 2 8 X¹E6^(c) 497/8 −TG 68   86^(c) 15 50 5 7 48  71^(c) 12 54 4 4 X E7^(d) 698 A→G 0  85 0 38 1 7 0 58 0 4 0 4 X¹ E9^(e)  970 G→A 0 110 10 52 0 60 79 4 52 0 7 X E10^(f) 1116 C→G 0 106 0 36 0 6 0 71 1 49 0 4 X E10^(g)1354 G→A 1 102 0 41 0 6 1 63 0 3 0 3 X E10^(h) 1456 A→G 0  91 0 40 0 6 158 0 4 0 3 X E10^(i) 1466 C→T 23 110 7 49 1 7 27 82 3 52 3 7 X E10^(j)1487 C→A 0  62 1 10 0 6 0 12 0 50 0 3 X

TABLE 18 Synonomous Variants Identified in CHRNA7 and dupCHRNA7Schizophrenics Controls Afr. Afr. Cauc. Am. Hisp. Cauc. Am. Hisp. MapSite bp change V T V T V T V T V T V T α7 dα7 E1 45 G→A 1 99 0 41 0 6 064 0 3 0 3 X E7 654 C→T 77 90 32 47 5 6 57 70 3 4 3 3 X E7 690 G→A 82 8336 36 6 6 59 59 4 4 3 3 X X E8 861 C→T 4 98 1 40 1 7 1 59 0 4 0 3 X E9921 G→A 2 112 1 45 0 6 4 77 0 50 0 7 X E9 933 G→A 56 127 28 53 6 8 39 7918 50 6 7 X A only 2 127 E9 966 C→T 1 110 6 46 0 6 0 79 2 52 0 7 X¹ E101044 C→T 12 123 3 43 0 6 9 72 1 55 1 5 X E10 1116 C→T 2 107 8 44 0 6 071 6 54 1 5 — — E10 1269 C→T 75 95 29 40 5 6 47 57 2 3 3 3 X X T only 295 1 40 1 57 E10 1335 C→T 32 74 3 11 2 7 30 65 2 4 1 3 X X

TABLE 19 Non-coding Variants Identified in CHRNA7 and dupCHRNA7Schizophrenics Controls Afr. Afr. Cauc. Am. Hisp. Cauc. Am. Hisp. MapSite bp change V T V T V T V T V T V T α7 dα7 I2 +75 G→A 0 87 1 38  0 61 50 0 3 0 1 X I2 +82 A→C 0 87 2 38  0 6 0 50 0 3 0 1 X I3 −9 A→G 0 1133 45  0 6 0 103 1 55 0 8 X I7 +21 C→T 21 31 1 6 1 3 3 3 0 0 0 0 — — I7−11 +GTT 10 38 2 4 0 4 5 10 0 1 0 1 — — I7 −20 G→A 15 37 1 4 2 4 5 10 11 1 1 — — I7 −29 T→G 1 37 0 4 0 4 0 10 0 1 0 1 — — I9 +19 C→T 0 43 0 5 07 1 78 4 54 0 7 — — I9 +27 −TCGGAG 0 110 1 44  0 6 0 78 2 54 0 7 — — I9+37 G→C 56 126 36 58^(k ) 6 8 38 79 17 53 6 7 — — 3′UT 1737 C→A 1 34 0 50 2 0 33 0 1 0 1 — — 3′UT 1837 T→G 0 34 1 6 0 2 0 33 0 1 0 1 — —

TABLE 20 Allele Frequencies of Rare Variants Amino Frequency FrequencyMap Site bp change Acid Ethnicity Schizo. Controls α7 dα7 E1 45 G→ACauc. 0.005 0 X I2 +75 G→A Cauc. 0 0.01 X I2 +82 A→C Afr. Am. 0.026 0 XI3 −9 A→G Afr. Am. 0.033 0.009 X E4 334 A→G I112V Afr. Am./Hisp. 0.0100.008* X E8 861 C→T Cauc. 0.020 0.008 X E9 921 G→A Cauc. 0.009 0.026 XE9 921 G→A Afr. Am. 0.011 0 X I9 +19 C→T Cauc. 0 0.006 — — I9 +27−TCGGAG Afr. Am. 0.011 0.019 — — E10 1116 C→G S372R Afr. Am. 0 0.010 — XE10 1354 G→A 452K Cauc. 0.005 0 X — E10 1456 A→G I486V Cauc. 0 0.009 X3′UT 1737 C→A Cauc. 0.015 0 — — *Variant 334 was found in 1/43 AfricanAmerican and 0/6 Hispanic schizophrenics, and 0/55 African American and1/8 Hispanic controls.

TABLE 21 Variants with Significantly Different Frequencies by EthnicityAfrican Caucasian American P Map Site bp change V T V T values α7 dα7 I3−9 A→G 0 216 4 100 0.0104 X E6 497/8 −TG 116 167 27 104 <0.0001 X E7 654C→T 134 160 35 51 <0.0001 X E9 966 C→T 1 189 8 98 0.0010 X E9 970 G→A 0189 14 104 <0.0001 X I9 +19 C→T 0 121 4 59 0.0475 — — I9 +27 −TCGGAG 0188 3 98 0.0394 — — E10 1116 C→T 2 178 14 98 <0.0001 — — E10 1466 C→T 50192 10 101 0.0011 X

Example 19 Mapping Variants to the CHRNA7 and dupCHRNA7 Genes

CHRNA7 exons 5-10 are duplicated and nearly homologous (>99%),complicating the mutation screen. However, the duplicated exons aretranscribed with different 5′ sequence and thus were isolated as uniquemRNA species. The cDNA primer sets, used to specifically amplifyfull-length cDNA from either CHRNA7 or its duplication (dupCHRNA7), arelisted as the last two entries in Table 16. These cDNA templates werethen used to map the variants in exons 5-10, using RT-PCR and subsequentSSCP and sequence analysis of the RT-PCR products.

Eighty-four samples from the mutation screening study were used for cDNAmapping of the eight common variants. Immortalized cell lines were notavailable from the NIMH Schizophrenia Initiative samples and, thus,postmortem brain and immortalized lymphoblasts collected locally in theDenver Schizophrenia Center were utilized.

Immortalized lymphocytes were cultured 6 hours with 1 mg/mlcyclohexamide before RNA isolation. Total RNA was isolated frompostmortem human hippocampus or cyclohexamide-treated immortalizedlymphocytes, using TRIzol reagent (Life Technologies, Gibco-BRL). RNAwas reverse transcribed (500 ng) using Superscript II reversetranscriptase components (Gibco-BRL) with 8 μM random hexamers(Pharmacia & Upjohn Diagnostics, Kalamazoo, Mich.) and 0.5 U placentalRNase inhibitor (Boehringer-Mannheim, Indianapolis, Ind.). A primary PCRwas performed using specific primers designed with Oligo software 4.1(National Biosciences, Inc., Plymouth Minn.). Full-length CHRNA7transcripts were amplified using 1 M GC-melt and 10× cDNA buffer(Clontech, K1905-1) from the Advantage cDNA PCR kit (CLONTECH,Laboratories Inc., Palo Alto, Calif.) and a two-step program withannealing temperatures from 72° C. to 68° C. Partially duplicateddupCHRNA7 transcripts were amplified using 1 M GC melt and 5× cDNAbuffer from the Advantage-GC cDNA PCR kit (K1907-1). These primaryreactions were then analyzed using SSCP and sequence analysis.

As shown herein, exons 5-10 of the α7 nicotinic receptor subunit geneare duplicated. Genomic variants in these exons, therefore, arecontemplated to be present in either the full-length CHRNA7 gene or indupCHRNA7. Polymorphisms were mapped, when possible, to one of the twoduplicons, utilizing mRNA isolated from either immortalized lymphoblastsor postmortem brain by using gene specific PCR. In some cases a givenvariant was present in both duplicons. In others, only tissue from aschizophrenic subject was available for mapping. In this case, the mapsite is indicated as provisional, since gene rearrangements orconversions could have occurred.

Eight of the more common variants were mapped in 32 samples fromindividuals with schizophrenia and 52 samples from control individuals(total of 84). Four common variants: the 497/8 2 bp deletion, theneutral variant at 654 bp, the neutral variant at 1044 bp, and the aminoacid changing variant at 1466 bp, all mapped only to dupCHRNA7 (See,Table 22). The 2 bp deletion in exon 6 was found in 15 out of the 32Caucasians with schizophrenia, 29 out of the 49 Caucasian controlsamples, 1 out of the 4 African Americans with schizophrenia, and 2 outof the 3 African American controls.

Three common neutral variants, at 690 bp, 1269 bp, and 1335 bp mapped toboth duplicons. The very common variant at 690 bp mapped primarily tothe duplicated gene (69 out of 72 individuals). The 1269 bp variantmapped to both CHRNA7 genes in 14 out of 54 individuals, while theneutral variant at 1335 bp mapped primarily to the full-length CHRNA7gene, and the variant at 933 bp mapped only to the full-length gene.Variant 933 bp G→A is in linkage disequilibrium with an intronicvariant, and is contemplated to involve splicing.

TABLE 22 Mapping of common variants, identified in the CHRNA7 gene andits partial duplication Schizophrenics Controls Exon/ Caucasian Af. Am.Caucasian Af. Am. In- genomic cDNA genomic cDNA genomic cDNA genomiccDNA Map tron bp Δ V T α7 dα7 T V T α7 dα7 T V T α7 dα7 T V T α7 dα7 Tα7 dα7 E-6^(a) 497-8 −TG 68 96 0 15 15 15 50 0 1 1 48 71 0 29 29 12 54 02 2 X E-7 654 C→T 77 90 0 20 20 32 47 0 1 1 57 70 0 33 33 3 4 0 1 1 XE-7 690 G→A 82 83 4 22 24 36 36 1 3 4 59 59 4 41 41 4 4 0 3 3 X X E-9933 G→A 56 127 12 0 12 28 53 3 0 3 39 79 15 0 15 18 50 1 0 1 X A only 2127 E-10 1044 C→T 12 123 0 3 3 3 43 0 1 1 9 72 0 3 3 1 55 0 0 0 X E-101269 C→T 75 95 9 13 18 29 40 2 3 4 47 57 18 22 31 2 3 0 1 1 X X T only 295 1 40 1 57 E-10 1335 C→T 32 74 6 2 7 3 11 0 0 0 30 65 17 3 19 2 4 2 02 X X E-10^(b) 1466 C→T 23 110 0 4 4 7 49 0 0 0 27 82 0 12 12 3 52 0 0 0X E, exon; I, intron. Numbering for exons and 3′UT is from the ATGstart. α7, full-length gene. dα7, duplicated gene. total, number ofindividuals with the variant that were mapped. ^(a)exon 6, 2 bp deletionat leu a.a. 166; ^(b)exon 10, ser to leu a.a. 489.

Ten of the thirty-three variants in Tables 17-19 were not mapped. Sevenof the unmapped variants lie in introns and could not be mapped usingthe cDNA specific RT-PCR methodology. One unmapped variant in exon 10was discovered late in the screen and was found to be synonymous.

A large number of variants (12) were found in a short proximal promoterregion 5′ of the translation start as shown in FIG. 16 panel A and aspublished (Leonard et al., Arch Gen Psychiatry, 59:1085-1096, 2002). Asdescribed herein, many of the variants were found to functionally reducetranscription in a reporter gene assay and to be associated with boththe P50 auditory gating deficit and with schizophrenia. Therelationships of these promoter polymorphisms to some of the variants inthe coding and non-coding sequence are discussed below.

Non-Synonymous Variants

The coding region of the full-length CHRNA7 gene consists of 10 exons.Eleven variants mapping to the full-length gene are reported in Tables17-19, three of which are non-synonymous. The A→G variant at 334 bp inexon 4, results in a conservative amino acid change of an isoleucine toa valine at amino acid 112. However, this residue lies in the putativeagonist-binding site (Galzi et al., Annu Rev Pharmacol, 31:37-72, 1991),where a conformational alteration is contemplated to result in a changein agonist affinity. The rare variant at 334 bp was found in one AfricanAmerican schizophrenic but not in an affected sibling and in oneHispanic control subject. The control subject exhibited abnormalauditory evoked potential responses, having a P50 test to conditioningratio of 1.91. Both subjects with this rare 334 bp variant also have arare insertion in the α7 core promoter (−190+G), indicating that thisrepresents a minor haplotype. The schizophrenic, however, also carries acore promoter mutation on the other chromosome (−178-G).

The G→A variant at 1354 bp in exon 10 changes a glutamic acid to alysine in the large intracellular loop of the protein. A glutamic acidat this position is conserved across species. In the rat, a largedeletion of sequence including this codon resulted in a two-foldincrease of both α-bungarotoxin binding and current in transfectedoocytes (Valor et al., Biochem, 41:7931-7938, 2002). However, the singlenon-conservative change from an acidic to a basic residue describedherein is expected to effect a functional change in the receptor. Therare variant at 1354 bp was found in one Caucasian schizophrenic and inone Caucasian control subject. Both of these subjects have normal corepromoter sequences. Although not having the 1354 bp variant, an affectedbrother of the schizophrenic has a mutation in the core α7 promoter (−86bp), indicative of two α7 alleles for schizophrenia in this family.

The C→A variant at base pair 1487 in exon 10 changes an alanine to anaspartic acid in the extracellular carboxyl terminus. The 1487 bpvariant was found in one African American schizophrenic but not in anaffected child. A family member with an abnormal P50 test toconditioning ratio of 61.7 carried an α7 core promoter mutation (−191G→A), again indicative of two alleles for schizophrenia.

Sixteen variants found in α7 exons 5-10 mapped to the duplicated genedupCHRNA7, which is also in the region of chromosome 15q14 geneticallylinked to schizophrenia (Tables 17-19 and FIG. 16 panel A). The mRNA fordupCHRNA7 is expressed in multiple tissues, including brain (Drebing etal., Soc Neurosci Abst, 24:832, 1998). DupCHRNA7 is present in only onecopy in approximately 30% of the general population, but ishomozygotically deleted in 5% of schizophrenic subjects (Gault et al.,Genomics 52: 173-185, 1998; and Leonard et al., Biol Psych 49:571,2001). Recent evidence suggests that dupCHRNA7 transcripts aretranslated, but the function of this protein is not yet known (Lee etal., Soc Neurosci Abst, 27:144.10, 2001). Six single nucleotidepolymorphisms (SNP) change amino acids in a putative open reading framefound in dupCHRNA7 (370 bp in exon 5, 698 bp in exon 7, 970 bp in exon9, and 1116 bp, 1456 bp, and 1466 bp in exon 10).

A 2 bp deletion at bases 497/8 in exon 6 was found in one copy of theduplicated gene in 57.5% of schizophrenic subjects and in 49.6% ofcontrols (not a significant difference). It was, however, found morefrequently in Caucasian control subjects than in African Americancontrols (X²=25.31, p<0.0001). This deletion, found only in dupCHRNA7,shifts the reading frame, resulting in three stop codons within the next53 codons. These stop codons, however, are the most frequently skippedduring translation (MacBeath and Kast, BioTechniques, 24:789-794, 1998).Further, the site surrounding the deletion in exon 6 is a consensus exonsplice enhancer site (ESE) for enhancer factor SC35 (Cartegni et al.,Nat Rev Gen, 3:285-298, 2002). Deletion of the two base pairs (TG) iscontemplated to disrupt this site, indicating that exon 6 is spliced outin these subjects, leaving an exon 5/exon 7 junction. This splicevariant would leave the coding sequence in frame. Deletion of exon 6removes the cysteine bridge and part of a putative ligand binding site,leaving the remainder of the α7 coding sequence intact. In the analysisof the CHRNA7 proximal promoter described herein, subjects with apromoter variant were much less likely to have a 2 bp deletion in exon 6of the dupCHRNA7 gene (X²=16.46, 1; p<0.0001). There was also a strikingrelationship with a three base pair insertion in intron 7. Every subject(50 out of 50) with a 2 bp deletion in exon 6 of the dupCHRNA7 gene,also had this insertion (+GTT) at the −11 bp position in intron 7. Thisintronic variant is contemplated to reside in the gene duplicationrather than in the full-length gene.

Synonymous Variants

Eleven SNPs in the coding regions that do not result in an amino acidchange were found. Four conservative exon variants at bp 690, 1269, and1335 map to both the duplicated gene and the full-length CHRNA7 gene.The variant at 690 bp in exon 7 is the most common variant found in theα7 nicotinic receptor genes, and it is heterozygous in genomic DNA from190 of 191 samples examined. The G primarily maps to CHRNA7 and the Aprimarily maps to dupCHRNA7. The 1269 bp and 1335 bp variants were foundin 80% and 43% of all subjects, respectively.

Another common synonymous variant in exon 9, at bp 933, is of interest.It was found only in the full-length gene and is also inverselyassociated with the presence of a polymorphism in the proximal promoterin all subjects examined, X²=6.916, 1; p=0.0085. The association wassignificant in the controls (X²=5.183, 1; p=0.0228), but only suggestivein the schizophrenic subjects. The 933 G→C variant is found within theloop of a putative stem and loop structure formed by a tri-nucleotiderepeat of (GGT)₃ and its complement repeat (ACC)₃ in exon 9 (ΔG=−16.2kcal/mol). The 933 bp variant is also in linkage disequilibrium with acommon intronic variant in intron 9 as discussed below.

Intronic Variants

Ten intron changes were identified, none of which change the consensussequences at RNA splice junctions. However, a number of these variantsmay affect splicing by introducing a favorable splice site or affectingthe binding sites of splice enhancer proteins. In intron 3, a variant at−9 (G→A) changes the sequence near the 3′ acceptor site to a sequenceidentical to nine bp in exon 4, thereby forming a cryptic splice site.Although found in only 3 of 45 African American schizophrenic families(3/90 alleles), this polymorphism was found in only 1 of 55 AfricanAmerican controls (1/110 alleles). The single control subject with thisvariant had a P50 (test to conditioning ratio) of 0.32, in the unstablerange, and had been diagnosed with major depression.

The intron 7 variant at −11 (+GTT) was mentioned above in relation tothe 2 bp deletion in exon 6. Insertion of these three base pairsintroduces additional pyrimidines into the splice acceptor site for exon7, possibly increasing site use. Another intron 7 variant at −20 (G→A),is inversely associated with the presence of proximal promoter variants.Only 1 of 29 subjects with the polymorphism had a promoter mutation,while 20 of 58 subjects with the wild-type sequence had a promoterpolymorphism (X²=10.17, 1; p=0.0014).

A variant in intron 9 (+37, counted from the splice donor site) wasfound more frequently in the African American schizophrenic sample thanin the control sample (X²=9.986, 1; P=0.0016). This same variant was notfound at significantly different frequencies in the Caucasianschizophrenia sample. One unaffected family member was identified with ahomozygous C at base pair +37. Interestingly, the exon 9 variant at 933bp (G→A) is in linkage disequilibrium with the intron 9 variant at +37(G→C). Since the exon 9 variant at 933 appears to be in the full-lengthgene, it is contemplated that the intron 9 variant is also located inthe full-length gene. If the intron 9 variant at +37 is not associatedwith the exon 9 variant at 933, then there is contemplated to be anotherpolymorphism present nearby (e.g., exon 9 variant at 966, C→T), which isalso present in the full-length gene. However, the exon 9 variant at 966is rare and was only mapped in one individual who was a schizophrenic,and thus its map location is provisional.

Example 20 Statistical Analysis of CHRNA7 and dupCHRNA7 Variants

Chi square statistics or Fisher's exact tests were used to determinewhether a variant was found more frequently in the schizophrenic samplethan the control sample. Allele frequencies were calculated for variantsin exons 1-4, but could not be determined for polymorphisms in theduplicated exons. A case-control study was done. All schizophrenicsubjects in each family were screened for polymorphisms to determine ifvariants cosegregate with affected family members and to ensure that nomutations were missed. Total counts from schizophrenic individualsinclude one schizophrenic individual from each family, unless otherschizophrenic family members differed from the proband at thatnucleotide position. When this occurred, the other family member wasalso counted. The sample size provided sufficient power to detect a 0.11difference in allele frequency between the schizophrenic and controlgroups at a p<0.05 for an allele with a population frequency of 0.050.

Two population-specific loci, FY-null and RB2300, were used to estimatethe degree of admixture in African American samples of schizophrenicindividuals and controls (Parra et al., Am J Hum Gen, 63:1839-1851,1998). The FY-NULL*1 allele is the normal allele with a C at −46 in thepromoter of the DARC gene (Duffy antigen receptor of chemokines). The FYNULL*1 allele has an allele frequency of 1.0 in European populations, 0in African populations and 0.06-0.2 in African American populations(Parra et al., supra, 1998). FY-NULL*1 allele frequencies did not varysignificantly between the African American controls (0.2) and theschizophrenic individuals (0.18) studied herein. The RB2300*1 allele hasan allele frequency of 0.900 to 0.944 in African populations, 0.776 to0.888 in African American populations, and 0.287 to 0.588 in Europeanpopulations (Parra et al., supra, 1998). The RB2300*1 allele does nothave a BamHI polymorphism in intron 1 of the human retinoblastoma gene.The RB2300*1 allele was found at a frequency of 0.82 in our AfricanAmerican controls and 0.86 in the African American subjects withschizophrenia (not significantly different). These data suggest thatthere is a similar degree of admixture in our African American controland schizophrenic samples and that differences in variant frequenciesbetween these samples should not reflect ethnic bias.

Example 21 Identification and Analysis of Distal Promoter PolymorphismsSubjects

Characteristics of study participants are shown below. See, Table 28 andTable 29.

TABLE 28 Number of individuals for case-control and family-basedassociation studies with the a priori outcome of schizophrenia TotalCase-control Denver SZ NIMH SZ COS Total SZ controls African-American 5647 2 105 45 Caucasian 234 73 56 363 99 NIMH Families SchizophrenicsFamily Members African-American 113 32 Caucasian 14242 Denver SZ =schizophrenics collected locally in Denver. NIMHSZ = schizophrenicindividuals from the NIMH Schizophrenia Genetics Initiative included incase control association studies. COS = Childhood onset schizophrenicscollected in Denver. Family Members = NIMH family members with adiagnosis of never mentally ill.

TABLE 29 Number of individuals for case-control and family-basedassociation studies with the outcome of smoking status Denver NIMH TotalDenver NIMH Total Case-control Sm Sm Sm NS NS NS African-American 54 559 49 15 64 Caucasian 160 10 170 112 13 125 NIMH Families Smokers (Sm)Non-Smokers (NS) African-American 17 45 Caucasian 25 51 Sm = smokers, NS= non-smokers.

DNA samples from both case-control subjects collected in Denver, andschizophrenic subjects from NIMH families were included in theassociation studies. For the family-based study, a total of 329African-Americans (47 nuclear families) and Caucasian-Non Hispanicsubjects (73 nuclear families) from the NIMH Schizophrenia GeneticsInitiative were chosen based on a diagnosis of schizophrenia. Thiscohort has previously shown positive LOD scores and association tomarkers at the 15q14 locus in both African-American and Caucasianindividuals. Kaufmann et al., “NIMH Genetics Initiative MilleniumSchizophrenia Consortium: linkage analysis of African-Americanpedigrees” Am. J. Med. Genet. 81:282-289 (1998); Leonard et al.,“Further investigation of a chromosome 15 locus in schizophrenia:analysis of affected sibpairs from the NIMH Genetics Initiative” Am. J.Med. Genet. 81:308-312 (1998); and Freedman et al., “Genetic linkage toschizophrenia at chromosome 15q14” Am. J. Med. Genet. 105: 655-657(2001). Detailed information on the NIMH family structure is availablefrom the web site nimhgenetics.org.

Case-control association studies of 612 African-American andCaucasian-Non Hispanic schizophrenics and controls included individualscollected in our laboratory, and schizophrenic subjects from the NIMHfamilies. Ethnicities of case-control subjects were recorded fromself-report or family interview. The Caucasian-Non Hispanic case-controlsample was comprised of 307 schizophrenic patients, 56 childhood onsetschizophrenics, and 99 controls. The African-American case-controlsample included 103 schizophrenics, 2 childhood onset schizophrenics,and 45 controls. P50 data were available for 89 Caucasian-Non Hispaniccontrols subjects and 43 African-American controls.

Adult case subjects were chosen based on a diagnosis of schizophrenia,utilizing a Structured Clinical Interview for DSM-IV Axis I Disorders.First et al., “Structured Clinical Interview for Axis I DSM-IVDisorders—Patient Edition (With Psychotic Screen)—(SCID-I/P, W/PSYCHOTICSCREEN) (Version 2.0)” Biometrics Research Department, New York StatePsychiatric Institute, New York (1996). Control subjects wereinterviewed and found to have no evidence for current or past psychosis,using a Structured Clinical Interview for DSM-IV Axis I Disorders fornon-patients. First et al., “Structured Clinical Interview for Axis IDSM-IV Disorders—Non-patient Edition—(SCID-I/NP, Version 2.0)”Biometrics Research Department, New York State Psychiatric Institute,New York (1996). Childhood onset schizophrenia cases were diagnosedutilizing the K-SADS-PL. (Kaufman et al., “Schedule for affectivedisorders and schizophrenia for school-age children present and lifetimeversion (K-SADS-PL): initial reliability and validity data” J. Am. Acad.Child Adolesc. Psych. 36: 980-988 (1997). Smoking history was determinedon local subjects utilizing a detailed questionnaire. Breese et al.,“Effect of smoking history on [3H]nicotine binding in human postmortembrain” J. Pharmacol. Exp. Ther. 282:7-13 (1997); Breese et al.,“Abnormal regulation of high affinity nicotinic 614 receptors insubjects with schizophrenia” Neuropsychopharmacology 23:615:351-364(2000). NIMH Genetics Initiative subjects utilized the questionnairefrom the NIMH web site (nimhgenetics.org). P50 auditory evokedpotentials were recorded on controls. Freedman et al., “Elementaryneuronal dysfunctions in schizophrenia” Schizophr. Res. 4:233-243(1991).

DNA Isolation

Nucleated cells were obtained from anticoagulated blood (EDTA) via lysiswith a high sucrose solution (0.3 M sucrose) were not associated witheither schizophrenia or the P50 112340123456789 gating deficit. Gault etal., “Comparison of polymorphisms in the α7 nicotinic receptor gene andits partial duplication in schizophrenic and control subjects” Am. J.Med. Genet. 123B:39-49 (2003).

Mutation screening of the core promoter in the CHRNA7 gene identified alarge number of polymorphisms. Functional analysis of polymorphisms inthe 231 base pairs upstream of the translation initiation site, the corepromoter region, demonstrated that most decrease transcription. Thesepolymorphisms were also found to be statistically more prevalent inschizophrenics than in control subjects (P=0.007). Further, the presenceof a promoter polymorphism in non-schizophrenic controls was associatedwith a P50 gating deficit (P<0.0001). Leonard et al., “Association ofpromoter variants in the alpha 7 nicotinic acetylcholine receptorsubunit gene with an inhibitory deficit found in schizophrenia” Arch.Gen. Psychiatry 59:1085-1096 (2002).

Reporter gene assays with fragments 1.0 kb and 2.6 kb proximal to thetranslation initiation site exhibit less activity than the corepromoter, suggesting that repressor elements may lie upstream of thecore promoter. Utilizing overlapping genomic fragments, 35 SNPs havebeen identified in the 2 kb upstream regulatory region of CHRNA7. SNPswere genotyped utilizing a combination of heteroduplexanalysis bydenaturing high-performance liquid chromatography and sequencing, andwere analyzed for association with schizophrenia in Caucasian-NonHispanic and African-American subjects. Smoking history was consideredas a secondary outcome. The results show significant association of aspecific SNP, rs3087454 (−1831 bp), in the 5′ upstream regulatory regionof the CHRNA7 gene with schizophrenia.

Genotyping and Fragment Analysis

Eleven amplicons were designed to screen 2.0 kb of DNA in the5′-upstream regulatory region of the CHRNA7 gene. The Polymerase ChainReaction (PCR) with AmpliTaq Gold™ and GeneAmp® PCR System 9600(Perkin-Elmer, Foster City, Calif.) was used for fragment amplificationwherein thermal cycler conditions were unique to each amplicon. See,FIG. 20.

Fragments were characterized using temperature modulated heteroduplexanalysis with Denaturing High-Performance Liquid Chromatography (DHPLC;Transgenomic WAVE™) (Transgenomic Inc., San Jose, Calif.) as previouslydescribed. Leonard et al., “Association of promoter variants in thealpha 7 nicotinic acetylcholine receptor subunit gene with an inhibitorydeficit found in schizophrenia” Arch. Gen. Psychiatry 59:1085-1096(2002); and Gault et al., “Comparison of polymorphisms in the α7nicotinic receptor gene and its partial duplication in schizophrenic andcontrol subjects” Am. J. Med. Genet. 123B:39-49 (2003). Samples were“spiked” with control DNA to ensure that a homozygous variant that mightmigrate as a single peak would be detected. When a sample resulted in anunusual WAVE™ pattern, automated DNA sequencing on an Applied Biosystems3100 Avant DNA Sequencer (Applied Biosystems, Foster City, Calif.) wasemployed for genotyping. For most SNPs, a specific and recognizableWAVE™ pattern was generated.

Cell Culture

The human neuroblastoma cell lines SH-SY5Y (a gift from Dr. JuneBiedler); Biedler et al., “Multiple neurotransmitter synthesis by humanneuroblastoma cell lines and clones” Cancer Res. 38:3751-3757. (1978),and SK-N-BE (ATCC number CRL-271; ATCC, Manassas, Va.) were grown in 1:1Ham F12 Dulbecco Eagle medium supplemented with 15% fetal bovine serum,100 μg/ml streptomycin, 100 units/ml penicillin, and 2 mM L-glutamine(Invitrogen, Carlsbad, Calif.) at 37° C. in a 5% CO₂ incubator.

Plasmid Constructs

Isolation and characterization of 2.6 kb of 5′-flanking sequence in thehuman CHRNA7 subunit gene was described previously. Gault et al.,“Genomic organization and partial duplication of the human α7 neuronalnicotinic acetylcholine receptor gene” Genomics 52:173-185 (1998);Leonard et al., “Association of promoter variants in the alpha 7nicotinic acetylcholine receptor subunit gene with an inhibitory deficitfound in schizophrenia” Arch. Gen. Psychiatry 59:1085-1096 (2002).

The promoter-luciferase construct Pr2B contains a CHRNA7 5′-upstreamregulatory region fragment from −2004 bp to the translation start siteat +1 bp inserted upstream of the luciferase gene in the pGL3-Basicvector. Pr2B was generated by first TA cloning a 2004 bp PCR fragmentfrom the previously cloned 2.6 kb promoter into pDrive, according tomanufacturer's instructions (Promega, Madison, Wis.). Restrictionenzymes (HindIII and KpnI) were then used to isolate the 2004 bpfragment and insert it into the pGL3 reporter plasmid. Thepromoter-luciferase fusions were confirmed by DNA sequencing. Pr2Bcontains the (C) allele at −1831 bp. The −1831 (A) polymorphism wasintroduced into Pr2B by PCR, utilizing the QuikChange II XLSite-Directed Mutagenesis Kit, according to the manufacturer's protocols(Stratagene). The mutagenesis primers were: α7Prom-1831A forward(5′-gccatacatactccagaaaaaatAaataaattcccttggccc-3′) (SEQ ID NO: 182) andα7Prom-1831A reverse (5′-gggccaagggaatttattTattttttctggagtatgtatggc-3′)(SEQ ID NO: 183); the mutant construct was confirmed by DNA sequencingand named Pr2B-1831A.

Transient Transfection and Reporter Gene Assay

SH-SY5Y and SK-N-BE cells grown to subconfluency were transfected withthe ProFection Mammalian Transfection Calcium Phosphate System(Promega). A pGL3-Control construct in which the luciferase gene isregulated by the SV40 promoter and enhancer was used as a positivecontrol to measure the maximum reporter gene activity. A pRL-TKconstruct (Promega) with the Renilla luciferase gene, controlled by theHSV thymidine kinase promoter, served as an internal control fornormalization of transfection efficiency.

2×10⁵ cells/35 mm plate were incubated with molar equivalents of Pr2B,Pr2B-1831A, pGL3-Control, or pGL3-Basic luciferase reporter constructsand 1 μg of pRL-TK vector as an internal control for the transfectionefficiency. Cells were harvested after 48 h and luciferase activity wasdetermined with the Dual-Luciferase Reporter Assay System according tothe manufacturer's instructions (Promega). Each transfection wasperformed using eight separate plates in triplicate.

Statistical Analysis

1. Case-Control Data

The case group of schizophrenic individuals included one randomly chosenschizophrenic individual per family. For each marker, allele frequencieswere estimated and conformance of genotype frequencies withHardy-Weinberg Equilibrium (HWE) expected proportions was tested via a 1df chi-squared goodness-of-fit test using Haploview. Barrett et al.,“Haploview: analysis and visualization of LD and haplotype maps”Bioinformatics 21, 263-265 (2005).

Linkage disequilibrium parameters D′ and r2 were calculated for eachpair of markers and tests for differences in allele frequencies betweencases and controls were computed using a chi-squared test (1 df) foreach SNP in Haploview. Since several of the SNPs were rare, empiricalP-values were utilized rather than relying on the large-sampleapproximation. Further, since several SNPs were tested for each outcome,empirical P-values were generated via permutation that were correctedfor the number of SNPs tested. For each permutation, the minimum P-valueover all SNPs was compared to the minimum P-value over all SNPs in theoriginal data.

Chi-squared tests of association (2 df) between each SNP andschizophrenia under a genotypic model were calculated using the UNPHASEDsoftware version 3.0.13; a similar permutation procedure was applied.Dudbridge, F., “Pedigree disequilibrium tests for multilocus haplotypes”Genet. Epidemiol 25:115-121 (2003).

Corrected P-values are indicated with an (*). Odds ratio estimates (OR)and 95% confidence intervals (CI) for comparing relevant genotype orallele classes were computed with SPSS 16 301 software (SPSS Inc.,Chicago Ill.).

Analysis of the P50 gating deficit in controls was conducted usingsimple linear regression models that tested the mean differences in P50ratios under both additive and genotypic models of associationseparately for each SNP.

In the absence of evidence for ethnic-specific effects, ethnicities werecombined for most SNPs in order to gain power to detect association, andethnicity was included as a covariate.

All analyses were performed using SAS version 9.1 software (SASInstitute Inc.: Cary, N.C., 2004). Power estimates (at a significancelevel of 5%) for case-control analyses were obtained with the Quantosoftware. Gauderman, W. J., “Sample size requirements for associationstudies of gene-gene interaction” Am. J. Epidemiol. 155:478-484 (2002);and Gauderman, W. J., “Sample size requirements for matched case-controlstudies of gene-environment interaction” Stat. Med. 21:35-50 (2002).

Results indicated that the Caucasian schizophrenic/control sample sizehad power between 0.74 and 0.99 to detect a genotype relative risk(GRR)≧2.0 over a range of marker allele frequencies between 0.10 and0.40. At (GRR)≧2.0, power was substantially lower (0.12-0.88) for mostallele frequencies. The African-American schizophrenic/control samplesize had less power to detect a GRR≧2.0 (0.38-0.87) over a range ofmarker allele frequencies between 0.10 and 0.40. Similarly, at(GRR)≧2.0, power was considerably lower (0.07-0.49) for all allelefrequencies

2. Admixture Analysis

For both ethnicities, case-control samples were evaluated for thepresence of admixture by genotyping 176 ancestry-informative SNPmarkers. Hodgkinson et al., “Addictions biology:haplotype-based analysisfor 130 candidate genes on a single array. Alcohol. 43:505-515 (2008).Within each ethnic group, relative frequencies of alleles between thecases and controls were compared using a chi-squared test. Pritchard etal., “Use of unlinked genetic markers to detect populationstratification in association studies” Am. J. Hum. Genet. 65:220-228.(1999). There were no significant differences from what was expected,indicating a similar degree of admixture between cases and controls inboth ethnic groups. These conclusions were corroborated by estimatingindividual admixture proportions within each ethnic group usingstructure. Pritchard et al., “Inference of population structure usingmultilocus genotype data” Genetics 155:945-959 (2000). These analysesindicated no significant differences in average admixture proportionsbetween cases and controls within each ethnic group.

3. Family-Based Data

Prior to data analysis, PEDCHECK software was utilized to detect markerallele errors in Mendelian inheritance. O'Connell et al., “PedCheck: aprogram for identification of genotype incompatibilities in linkageanalysis” Am. J. Hum. Genet. 63:259-266 (1998). Pedigree DisequilibriumTest(s) (PDTs) of association at SNP markers were determined usingUNPHASED software version 3.0.13. Dudbridge, F., “Pedigreedisequilibrium tests for multilocus haplotypes” Genet. Epidemiol25:115-121 (2003). Similar to the case-controls, UNPHASED analysisgenerated an empirical P— value corrected for the number of SNPs.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention, which are obvious to those skilled inmolecular biology or related fields, are intended to be within the scopeof the following claims.

1. A composition comprising an isolated 5′ upstream regulatorynucleotide sequence of a human alpha-7 nicotinic receptor, wherein saidnucleotide sequence comprises at least one polymorphism.
 2. Thecomposition of claim 1, wherein said sequence comprises at least aportion of SEQ ID NO:181.
 3. The composition of claim 1, wherein said atleast one polymorphism comprises one or more of those listed in Table23.
 4. The composition of claim 1, wherein said polymorphism isrs3087454 (−1831 bp).
 5. A composition comprising an isolated regulatorybinding site sequence encoded by an isolated 5′ upstream regulatorynucleotide sequence of a human alpha-7 nicotinic receptor, wherein saidnucleotide sequence comprises at least one polymorphism.
 6. Thecomposition of claim 5, wherein said sequence comprises at least aportion of SEQ ID NO:181.
 7. The composition of claim 5, wherein said atleast one polymorphism comprises one or more of those listed in Table23.
 8. The composition of claim 5, wherein said polymorphism isrs3087454 (−1831 bp).
 9. A method, comprising: a) providing; i) anindividual suspected of having a predisposition to schizophrenia; ii) anucleic acid derived from said individual, wherein said nucleic acidcomprises an α7 nicotinic acid receptor regulatory allele; b) detectingat least one polymorphism within said α7 regulatory allele; and c)correlating the presence of said at least one polymorphism with apredisposition to schizophrenia.
 10. The method of claim 9, wherein saidat least one polymorphism comprises one or more of those listed in Table23.
 11. The method of claim 9, wherein said polymorphism is rs3087454(−1831 bp).
 12. The method of claim 10, wherein said at least onepolymorphism comprises two or more polymorphisms.
 13. The method ofclaim 9, wherein said at least one polymorphism contributes to reducedα7 regulatory protein transcription.
 14. The method of claim 9, whereinsaid detecting comprises at least one technique selected from the groupconsisting of polymerase chain reaction, heteroduplex analysis, singlestand conformational polymorphism analysis, denaturing high performanceliquid chromatography, ligase chain reaction, comparative genomehybridisation, Southern blotting, and sequencing.
 15. The method ofclaim 9, wherein said nucleic acid is derived from a biological sampleselected from the group consisting of a biopsy material and blood. 16.The method of claim 9, further comprising step (d) providing a diagnosisto said individual based on the presence or absence of the at least onepolymorphism.
 17. The method of claim 9, wherein said diagnosisdifferentiates schizophrenia from other forms of mental illness.
 18. Amethod, comprising: a) providing; i) a biological sample suspected ofcontaining a first polynucleotide encoding an α7 regulatory protein; ii)a second polynucleotide comprising at least a portion of SEQ ID NO: 181,capable of hybridizing to said first polynucleotide; b) hybridizing saidfirst polynucleotide to said second polynucleotide to produce ahybridization complex; c) detecting an α7 nicotinic acid regulatoryallele within said first polynucleotide, wherein said allele comprisesat least one polymorphism.
 19. The method of claim 18, wherein said atleast one polymorphism is rs3087454 (−1831 bp).
 20. The method of claim18, wherein at least one polymorphism comprises one or more of thoselisted in Table
 23. 21. The method of claim 18, wherein said methodfurther comprises step (d) detecting said hybridization complex, whereinsaid complex correlates with said first polynucleotide.
 22. The methodof claim 18, wherein said biological sample is selected from the groupconsisting of brain tissue and blood.
 23. The method of claim 18,wherein said biological sample is from a human.
 24. The method of claim23, wherein said human is suspected of suffering from a conditionselected from the group consisting of schizophrenia, small cell lungcarcinoma, breast cancer, and nicotine-dependent illness.
 25. The methodof claim 18, wherein said method further comprises, before step (b)amplifying said first polynucleotide by polymerase chain reaction.